Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use

ABSTRACT

The invention relates to novel variants of cytochrome P450 oxygenases. These variants have at least one mutation improving their ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. The variants also have at least one mutation improving thermostability as compared to the parent enzyme or corresponding wild-type enzyme. Preferred variants include cytochrome P450 BM-3 heme domain variants having L52I, I58V, F87A, H100R, S106R, F107L, A135S, M145A/V, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and/or V446I amino acid substitutions.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to variants of cytochrome P450 oxygenases.Specifically, the invention relates to thermostable variants ofcytochrome P450 oxygenases capable of improved peroxide-drivenhydroxylation, and methods of making thermostable variants.

2. Background Information

One of the great challenges of contemporary catalysis is the controlledoxidation of hydrocarbons. Processes for controlled, stereo- andregioselective oxidation of hydrocarbon feed stocks to more valuable anduseful products such as alcohols, ketones, acids, and peroxides wouldhave a major impact on the chemical and pharmaceutical industries.However, selective oxyfunctionalization of hydrocarbons remains one ofthe great challenges for contemporary chemistry. Despite decades ofeffort, including recent advances, the insertion of oxygen intounactivated carbon-hydrogen bonds (hydroxylation) remains difficult toachieve with high selectivity and high yield. Many chemical methods forhydroxylation require severe conditions of temperature or pressure, andthe reactions are prone to over-oxidation, producing a range ofproducts, many of which are not desired.

Enzymes are an attractive alternative to chemical catalysts. Inparticular, mono-oxygenases have unique properties that distinguish themfrom most chemical catalysts. Most impressive is their ability tocatalyze the specific hydroxylation of non-activated C—H, one of themost useful biotransformation reactions, which is often difficult toachieve by chemical means, especially in water, at room temperature andatmospheric pressure. These cofactor-dependent oxidative enzymes havemultiple domains and function via complex electron transfer mechanismsto transport a reduction equivalent to the catalytic heme center.

Cytochrome P450 monooxygenases (“P450s”) are a group ofwidely-distributed heme-containing enzymes that insert one oxygen atomfrom diatomic oxygen into a diverse range of hydrophobic substrates,often with high regio- and stereoselectivity. The second oxygen atom isreduced to H₂O. The active sites of all cytochrome P450s contain an ironprotoporphyrin IX with cysteinate as the fifth ligand, and the finalcoordination site is left to bind and activate molecular oxygen. Theirability to catalyze these reactions with high specificity andselectivity makes P450s attractive catalysts for chemical synthesis andother applications, including oxidation chemistry, and for many of theP450-catalyzed reactions, no chemical catalysts come close inperformance. These enzymes are able to selectively hydroxylate a widerange of compounds, including fatty acids, aromatic compounds, alkanes,alkenes, and natural products. Unfortunately, P450s are generallylimited by low turnover rates, and they generally require an expensivecofactor, NADH or NADPH, and at least one electron transfer partnerprotein (reductase). Furthermore, the enzymes are large, complex, andexpensive.

Wild-type P450s are in some cases capable of using peroxides as a sourceof oxygen and electrons via a peroxide “shunt” pathway, though theefficiency of this route is low. This secondary mechanism for substrateoxidation offers the opportunity to take advantage of P450 catalysiswithout the need for a cofactor, and eliminates the rate-limitingelectron transfer step carried out by the reductase. However, lowefficiency is a major limitation. Further, wild-type enzymes capable ofperoxide-driven hydroxylation, such as chloroperoxidase (CPO) andCYP152B1 are generally limited in their substrate specificity tohydroxylation of activated C—H bond carbons, i.e., carbon atoms adjacentto a functional group such as an aromatic ring, a carbonyl group, aheteroatom, etc.

One particular P450 enzyme, cytochrome P450 BM-3 from Bacillusmegaterium (“P450 BM-3”; EC 1.14.14.1) also known as CYP102, is awater-soluble, catalytically self-sufficient P450 containing a heme(monooxygenase/hydroxylase) domain which is 472 amino acids in lengthand a reductase domain that is 585 amino acids in length. The totallength of the enzyme is 1048 amino acids. The heme domain is generallyconsidered to end at position 472 and it is followed by a short linkerbefore the reductase domain begins. Because of the presence of anindependent reductase domain within the protein itself, P450 BM-3 doesnot require an additional or extraneous reductase for activity, but itdoes require an electron source, such as the cofactor nicotinamideadenine dinucleotide phosphate (NADPH). Nucleotide and amino acidsequences for P450 BM-3 are provided in FIGS. 1 and 2, respectively,which are the sequences for P450 BM-3 from the GenBank database,accession nos. J04832 (SEQ ID NO:1) and P14779 (SEQ ID NO:2),respectively.

P450 BM-3 hydroxylates fatty acids with a chain length between C12 andC18 at subterminal positions, and the regioselectivity of oxygeninsertion depends on the chain length. The optimal chain length ofsaturated fatty acids for P450 BM-3 is 14-16 carbons. P450 BM-3 is alsoknown to hydroxylate the corresponding fatty acid amides and alcoholsand forms epoxides from unsaturated fatty acids. The minimumrequirements for activity are substrate, diatomic oxygen, and thecofactor NADPH.

It has been demonstrated that ω-para-nitrophenoxycarboxylic acids(pNCAs) can be used as surrogate substrates for BM-3. When thissubstrate is hydroxylated at the ω position to produce ω-oxycarboxylicacid, the yellow chromophore p-nitrophenolate (pNP) is produced,allowing for easy detection of activity when screening mutant libraries.

Mutant P450 BM-3 enzymes with modified activity have now been reportedin the literature. For example, an F87A mutant was found to display ahigher activity for the 12-pNCA substrate, and, under NADPH-drivencatalysis, resulted in complete terminal hydroxylation of 12-pNCA,whereas the wild-type enzyme stopped at about 33% conversion. It hasalso been reported that the F87A mutant has a higher stability in H₂O₂solutions. (The convention in the art, which is adopted herein, is torefer to a mutant with reference to the native amino acid residue at aposition in the sequence, followed by the amino acid at that position inthe mutant, e.g., F87 refers to the phenylalanine at position 87 in thewild-type sequence, and F87A refers to the phenylalanine at position 87in the wild-type sequence which has been changed to alanine in thevariant. The numbering of the amino acid residues starts with the aminoacid residue following the initial methionine residue). It has beenshown that H₂O₂-driven hydroxylation to be much faster with the F87Amutation, as well as with an F87G mutation.

Powerful techniques for creating enzymes with modified or improvedproperties are now available, such as directed evolution (Arnold, 1998),in which iterative cycles of random mutagenesis, recombination andfunctional screening for improved enzymes accumulate the mutations thatconfer the desired properties. For example, mutants of cytochromeP450_(cam) or P450 BM-3 that hydroxylate the activated C—H bonds ofnaphthalene or 12-pNCA substrate, respectively, in the absence ofco-factors through the “peroxide-shunt” pathway, herein termed“peroxygenases,” have been created and identified using such techniques.In addition, P450 BM-3 mutants that can hydroxylate a variety ofnonnatural substrates, including octane, several aromatic compounds andheterocyclic compounds have been reported.

While the activity of enzymes has thus been improved and modified, acontinuing problem is that enzymes are often poorly stable underconditions encountered during production, storage or use. For example,improving enzyme resistance to thermal denaturation has been a majorfocus of protein engineering efforts. Improved thermostability oftencorrelates with longer shelf-life, longer life-time during use (even atlow temperatures), and a higher temperature optimum for activity.Stabilizing the relatively unstable cytochrome P450 enzymes by proteinengineering is a particularly challenging problem, however, partlybecause the P450s comprise multiple subunits and contain thermolabileco-factors. Two thermostable cytochrome P450s (CYP119 and CYP175A1) fromthermophilic organisms have recently been described and their (hemedomain) crystal structures determined. CYP119 exhibits a meltingtemperature of ˜91° C. Aromatic stacking, salt-link networks andshortened loops are believed to help stabilize these enzymes.Unfortunately, the functions of these P450s are not known, and reportedactivities are low (e.g., 0.35 min⁻¹ in the NADH-driven hydroxylation oflauric acid. While the International Patent application published as WO02/083868 found that the mutations M145A, L324I, I366V, and E442K in theP450 BM-3 heme domain promoted thermostability, the overallthermostability of the peroxygenase mutant was not higher than that ofthe wild-type heme domain.

Thus, there is a need in the art for useful oxidation catalysts whichare stable and do not require expensive cofactors or coenzymes forefficient oxidation and for methods of preparing the same. Thisinvention addresses these and other needs in the art.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of P450 BM-3mutations improving the thermostability of variants that have asignificantly improved ability to use peroxide as an oxygen source.

Thus, the invention provides an isolated variant of a cytochrome P450BM-3 comprising the amino acid sequence of SEQ ID NO:3, the variantcomprising at least a first mutation in an amino acid residue selectedfrom K9, I58, F87, E93, H100, F107, K113, A135, M145, 145A, A184, N186,D217, M237, E244, S274, L324, 1366, K434, E442, and V446 of SEQ ID NO:3,and at least a second mutation in an amino acid residue selected fromL52, S106, N239, and V340.

The invention also provides a method of thermostabilizing a variant of awild-type cytochrome P450 oxygenase heme domain, the variant having amutation in a first amino acid residue, the method comprising: preparinga protein library of variants of the parent having an additionalmutation in a second amino acid residue, which second amino acid islocated no more than 10 Ångströms from the first amino acid in thewild-type enzyme, and selecting any variant having a higherthermostability than the parent.

The invention also provides a variant of a wild-type cytochrome P450oxygenase heme domain comprising a mutation in a first amino acidresidue, which mutation promotes a higher ability to utilize peroxide asan oxygen source for oxidation of a substrate than the wild-type enzyme,and an additional mutation in a second amino acid residue, which secondamino acid is located no more than 10 Ångströms from the first aminoacid in the wild-type enzyme, which variant has a higher thermostabilitythan the wild-type enzyme.

The above features and many other advantages of the invention willbecome better understood by reference to the following detaileddescription when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the nucleic acid sequence of cytochrome P450 BM-3,GenBank Accession No. J04832 (SEQ ID NO:1).

FIG. 2 shows the amino acid sequence of cytochrome P450 BM-3, GenBankAccession No. P14779 (SEQ ID NO:2).

FIG. 3 shows the pCWori+ vector used for expression of, e.g., wild-typeP450 BM-3, P450 variants, or heme domains of P450 variants.

FIGS. 4A and 4B shows the Sequence alignments of P450 BM-3 heme domainwith the heme domain of exemplary P450 enzymes listed in Table 2.

FIG. 5 shows the representative topology diagram of the heme domain ofP450s. Helices are represented by black bars, and the length of each ofthe bars is in approximate proportion to the length of the helix. Thestrands of β-sheets are shown with arrows. The strands are grouped bythe secondary structural elements which they comprise. The structuralelements are grouped into the α-helical-rich domain and the β-sheet-richdomain. The heme is shown by the square at the NH₂-terminal end of theL-helix. With only minor modifications, this topology diagram could beused for other P450s (Peterson et al., 1995).

FIG. 6 shows the ribbon drawing of the wild-type cytochrome P450 BM-3heme domain with conserved secondary structure elements labeled asdescribed in FIG. 5. (A) and (B) each show different views of the P450BM-3 heme domain, indicating the sites of various mutations describedherein. (C) Mutations acquired during evolution of peroxygenase activityand which appear in mutant 21B3 (See WO 02/083868 by Cirino et al.) areshown as black balls. Mutations acquired through further directedevolution of thermostability and which appear in mutant 5H6 are shown ingrey balls. The atomic coordinates of P450 BM-3 described in Li andPoulos (1994) were used to create this image with the free-ware programSwiss PDB Viewer (available via the ExPASy (Expert Protein AnalysisSystem) proteomics server of the Swiss Institute of Bioinformatics (SIB)website).

FIG. 7A to 7D shows the four residue positions where mutations acquiredduring directed evolution of thermostability (L52, S106, E442, and M145)lie adjacent to positions (in the heme domain structure) where mutationswere previously acquired during evolution of peroxygenase activity.

FIG. 8 shows the percentage of 450 nm CO-binding peak of cytochrome P450BM-3 heme domain, HWT (white square); heme domain of F87A mutant, HF87A(white circle); and 5H6 (black triangle), remaining after 10-minuteincubation at the indicated temperatures. For the holoenzyme, BWT (whitediamond), the percentage of initial NADPH-driven activity remainingafter 10-minute incubations is shown.

FIG. 9 shows the heat-inactivation of cytochrome P450 BM-3 holoenzymeBWT (white diamond) and peroxygenase mutants HF87A (white circle) and5H6 (black triangle), calculated as the percentage of activity remainingafter incubation at 57.5° C. for the indicated periods of time.Peroxygenase activity was measured for HF87A and 5H6, while NADPH-drivenactivity was measured for BWT.

DETAILED DESCRIPTION OF THE INVENTION

Mutations in certain amino acid residues or regions of a P450 enzymecan, as shown herein, thermostabilize or stabilize an enzyme or enzymemutant. In particular, peroxygenase variants according to the presentinvention are more stable or thermostable than previously describedperoxygenase mutants, i.e., mutants of P450 enzymes more capable ofusing hydrogen peroxide for substrate oxidation than the correspondingwild-type enzyme. While many peroxygenase mutants previously known inthe art can function efficiently without the reductase domain and arenot dependent on cofactor, they have often suffered from a lowerstability or thermostability than the wild-type enzyme. The presentinvention addresses this problem by providing P450 variants which retainor substantially retain the improved peroxide-driven activity of aperoxygenase mutant while preserving or improving thermostability ascompared to the wild-type enzyme or corresponding region (e.g., hemedomain) of the wild-type enzyme. For example, the 5H6 mutant describedin Example 5 is more stable than the wild-type enzyme as well as thewild-type heme domain, and also has a many-fold higher peroxygenaseactivity over both the wild-type heme domain and the prior art mutantF87A.

Preferred mutation sites in the thermostable peroxygenase variantsinclude those that correspond to L52, S106, A184, and V340 in the P450BM-3 heme region (SEQ ID NO:3). For non-P450 BM-3 enzymes, thecorresponding wild-type enzyme preferably has at least 30, 40, 50, 60,70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3,and the mutated amino acid residues align with one or more of L52, S106,A184, and V340 in SEQ ID NO:3. In one embodiment, the amino acidsubstitutions at the respective sites are L521, S106R, A184V, and V340M.In another embodiment, the variant further comprises mutations in aminoacid residues corresponding to I58, F87, H100, F107, A135, N239, S274,L324, 1366, K434, E442, and V446. Preferably, the corresponding aminoacid substitutions are 158V, F87A, H100R, F107L, A135S, N239H, S274T,L3241, 1366V, K434E, E442K, and V4461. In yet another embodiment, thethermostable peroxygenase variant further comprises a deletion of ahistidine residue in a C-terminal 6-residue His-tag. See, Tables 2A, 2B,and 3 below.

Also described herein is a method of thermostabilizing or stabilizing aP450 peroxygenase mutant, as well as thermostabilized or stabilizedperoxygenase mutants. This method is based, in part, on the discoverythat thermostabilizing mutations can be found in amino acid residuesclose to amino acid residues previously mutated to introduceperoxygenase activity. Preferably, the amino acids are adjacent; eitherin the amino acid sequence or in the 3-dimensional structure of thewild-type enzyme when folded, i.e., there is no other amino acidsubstantially in-between the two amino acid residues. In a preferredembodiment, in the wild-type enzyme, the amino acid in which thethermostabilizing mutation is introduced is within 15, preferably within10, and most preferably within 7 Ångströms of the amino acid in whichthe peroxygenase mutation is introduced. Optimally, the two amino acidresidues are within a 5-7 Ångström distance in the wild-type enzyme.

Exemplary pairs of adjacent amino acid residues include L52 and I58;S106 and F107; S274 and M145; and K434 and E442. (See FIG. 7). In anexemplary embodiment, a peroxygenase mutant comprising a mutation in anamino acid residue corresponding to at least one of I58, F107, S274, andK434 of SEQ ID NO:3 can be thermostabilized by introducing an additionalmutation in the amino acid residue corresponding to L52, S106, M145, andE442, respectively. Preferably, the amino acid substitutions promotingperoxygenase activity correpond to one or more of I58V, F107L, S274T,and K434E, and the thermostabilizing amino acid substitutions preferablycorrespond to one or more of L521, S106R, M145A, and E442K.

Accordingly, a peroxygenase mutant of a wild-type enzyme can bestabilized or thermostabilized by creating a library of variants of theperoxygenase mutant having mutations in amino acid residues within 15,preferably 10, and more preferably within 7 Ångströms from a previouslyintroduced mutation, and the resulting library screened forthermostability as described in the Examples. The order in which theperoxygenase and thermostabilizing mutation are introduced is notimportant. Thus, in an alternative embodiment, the thermostabilizingmutation can be introduced within 15, 10, or 7 Angstroms of a residue inwhich a mutation is known or believed to promote peroxygenase activitybefore the actual peroxygenase mutation is made. For example, a libraryof variants having a thermostabilizing mutation can be prepared in afirst step, and the postulated peroxygenase mutation subsequentlyintroduced into selected variants or the entire library. The library isthen screened for peroxygenase activity and/or thermostability,preferably a thermostability or stability comparable or higher than thanof the corresponding wild-type enzyme, and a higher peroxygenaseactivity than the corresponding wild-type enzyme.

The improved P450 BM-3 heme domain variants provided by the inventionare useful for hydroxylation and other oxidation reactions on a varietyof substrates, and in particular, substrates with alkyl chains, such asfatty acids, alkanes, long-chain alcohols and detergents. These BM3catalyzed reactions can proceed without cofactor, in the presence ofperoxide. The improved variants require lower concentrations of peroxideto achieve the same conversion, or require less time at a given peroxideconcentration to achieve the same conversion than the wild-type hemedomain. The use of a thermostable variant comprising the heme domainwithout the reductase domain allows more functional protein to be madeper unit volume of fermentation and therefore improves the efficiency ofenzyme production.

The use of P450 variants lacking the reductase provides importantadvantages during production of the catalyst (fermentation). Inparticular, the heme domain is not functional in the absence of itsreductase or peroxide. The expression of functional cytochrome P450 caninhibit the growth of E. coli cells. Expression is also likely to have adeleterious effect on other host cells as well, limiting the ability ofthe cells to be used to produce large amounts of catalyst. It istherefore very beneficial to be able to make a variant lacking thereductase domain, because such a protein has no activity in the absenceof peroxide, is not deleterious to the fermentation process and reducesthe host cell toxicity, the reduced size of the protein and concomitantmetabolic load for its production leads to higher expression in anyorganism, and the heme domain alone is more easily engineered to bestable, since only the heme domain and not the whole protein would haveto be stabilized. The host cells can therefore be grown to high densityand high P450 expression levels can be achieved.

Another major advantage of using a peroxygenase variant lacking thereductase domain is the lower susceptibility of the protein to damage byproteolysis (the linker between heme domain and reductase domain isknown to be highly susceptible to proteolytic cleavage) and otherdenaturants. The significance of these features of the variants of theinvention becomes evident during production and purification of thecatalysts, as well as during its application, for example, in a washingmachine or chemical reactor.

Applications for the variants of the present invention include their useas additives to a laundry detergent where the enzyme would serve tomodify the properties of surfactants in the detergent by catalyzing achemical reaction during the wash or rinse. Peroxide is often used inlaundry applications, and it can be used to drive the P450-catalyzedreaction. The chemical reaction would alter the properties, e.g.,solubility, of surfactants added to the detergent or of oily stains onclothing, making them easier to remove from the clothing. That theperoxide-dependent variant are also more stable or thermostable areespecially advantageous for preparing enzymes less sensitive tolong-term storage, and in such applications when elevated temperaturesare desired. Enzymes which are stable at elevated temperatures typicallyhave maximum activity at higher temperatures compared to less stablecounterparts.

Another application for the variants of the present invention is inchemical synthesis. The heme domain mutants described here can be usedwith inexpensive peroxide to catalyze the same transformations as theholoenzyme with molecular oxygen and NADPH, and the synthesis can, ifdesired, be conducted at a higher temperature to increase the reactionrate, if needed. A suitable system for chemical synthesis would involvethe slow addition of peroxide to a mixture containing enzyme andsubstrate, and allowing the chemical reaction to proceed at roomtemperature or higher. Organic solvents can be used to improve thesolubility of the substrate in the reaction mixture.

A particular advantage of using the P450 BM-3 variants of the inventionis that P450 BM-3 catalyzed oxidation is not restricted to activated C—Hbond carbons, i.e., carbon atoms adjacent to electron-rich groups(aromatics, heteroatoms, carbonyl groups, etc.). For example, infatty-acid oxidation, while a P450 enzyme, such as CYP152B1, is capableof peroxide-driven oxidation, it can only hydroxylate the alpha-carbon(the carbon adjacent to the acid carbonyl) (Matsunaga et al., 2000).Chloroperoxidase (CPO) is also capable of peroxide-driven hydroxylationon a variety of substrates, yet only at activated carbon positions (vanDeurzen et al., 1997). The P450 BM-3 enzymes of the invention arecapable of peroxide-driven hydroxylation of completely unactivated,carbon atoms in the substrate. In addition to having improvedperoxide-driven hydroxylation activity, the P450 BM-3 variants describedin the invention also demonstrate improved peroxide-driven epoxidationactivity, such as in the epoxidation of styrene to styrene oxide.

In all of the possible applications, the peroxide-driven chemistryoffers significant safety advantages over using molecular oxygen.Peroxide is comparatively inexpensive, is available in concentratedform, and does not pose the explosion hazard of enriched oxygen inindustrial settings. This is particularly important when the substrateis flammable or explosive, such as propane or alkenes in general.

The following defined terms are used throughout the presentspecification, and should be helpful in understanding the scope andpractice of the present invention.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

“Cytochrome P450 monooxygenase” or “P450 enzyme” means an enzyme in thesuperfamily of P450 haem-thiolate proteins, which are widely distributedin bacteria, fungi, plants and animals. The enzymes are involved inmetabolism of a plethora of both exogenous and endogenous compounds.Usually, they act as terminal oxidases in multicomponent electrontransfer chains, called here P450-containing monooxygenase systems. Theunique feature which defines whether an enzyme is a cytochrome P450enzyme is traditionally considered to be the characteristic absorptionmaximum (“Soret band”) near 450 nm observed upon binding of carbonmonoxide (CO) to the reduced form of the heme iron of the enzyme.Reactions catalyzed by cytochrome P450 enzymes include epoxidation,N-dealkylation, O-dealkylation, S-oxidation and hydroxylation. The mostcommon reaction catalyzed by P450 enzymes is the monooxygenase reaction,i.e., insertion of one atom of oxygen into a substrate while the otheroxygen atom is reduced to water.

“Heme domain” refers to an amino acid sequence within an oxygen carrierprotein, which sequence is capable of binding an iron-complexingstructure such as a porphyrin. Compounds of iron are typically complexedin a porphyrin (tetrapyrrole) ring that may differ in side chaincomposition. Heme groups can be the prosthetic groups of cytochromes andare found in most oxygen carrier proteins. Exemplary heme domainsinclude that of P450 BM-3 (P450_(BM-P)), SEQ ID NO:3, as well astruncated or mutated versions of these that retain the capability tobind the iron-complexing structure. The skilled artisan can readilyidentify the heme domain of a specific protein using methods known inthe art.

An “oxidation”, “oxidation reaction”, or “oxygenation reaction”, as usedherein, is a chemical or biochemical reaction involving the addition ofoxygen to a substrate, to form an oxygenated or oxidized substrate orproduct. An oxidation reaction is typically accompanied by a reductionreaction (hence the term “redox” reaction, for oxidation and reduction).A compound is “oxidized” when it loses electrons. A compound is“reduced” when it gains electrons. An oxidation reaction can also becalled an “electron transfer reaction” and encompass the loss or gain ofelectrons or protons from a substance. Non-limiting examples ofoxidation reactions include hydroxylation (e.g., RH+O₂+2H⁺+2e⁻?ROH+H₂O)and epoxidation (alkene+2H⁺+2e⁻→epoxyalkene+H₂O).

A “peroxygenase” is an enzyme capable of functioning as an H₂O₂-drivenhydroxylase, i.e., inserting an oxygen from the peroxide into itssubstrate. Peroxygenase reactions include, but are not limited to,hydroxylation and epoxidation. In the case of many P450 enzymes, a“peroxygenase” can be a heme domain operating via the peroxide shuntpathway, using H₂O₂ or another peroxide as an oxygen source, in theabsence of NADPH or other co-factor and/or a reductase domain. A“peroxygenase mutant” or “peroxygenase variant” as described herein is acytochrome P450 enzyme having at least one mutation resulting in ahigher peroxygenase activity than the corresponding wild-type parentenzyme.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean a range of up to 20%, preferably up to 10%,more preferably up to 5%, and more preferably still up to 1% of a givenvalue. Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value.

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds.

A “secondary structural element” is a 3-dimensional structure in aprotein or protein variant. These secondary structural elements areformed by segments of the amino acid sequence which fold to certainconformations. As used herein, secondary structural elements include the“α-helix” or “helix”, a rod-like structure wherein a polypeptide segmentis folded by twisting into a right handed screw stabilized byhydrogen-bonding; “beta-pleated sheets,” also termed “beta sheets” orsimply “β” herein, wherein different segments of a polypeptide sequencerun side by side, either parallel or anti-parallel; and the polypeptidesegments joining different helices and/or beta sheets, called “loops.”

An “enzyme” means any substance, preferably composed wholly or largelyof protein, that catalyzes or promotes, more or less specifically, oneor more chemical or biochemical reactions. The term “enzyme” can alsorefer to a catalytic polynucleotide (e.g., RNA or DNA).

A “native” or “wild-type” protein, enzyme, polynucleotide, gene, orcell, means a protein, enzyme, polynucleotide, gene, or cell that occursin nature.

A “parent” protein, enzyme, polynucleotide, gene, or cell, is anyprotein, enzyme, polynucleotide, gene, or cell, from which any otherprotein, enzyme, polynucleotide, gene, or cell, is derived or made,using any methods, tools or techniques, and whether or not the parent isitself native or mutant. A parent polynucleotide or gene encodes for aparent protein or enzyme.

A “mutant”, “variant” or “modified” protein, enzyme, polynucleotide,gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell,that has been altered or derived, or is in some way different orchanged, from a parent protein, enzyme, polynucleotide, gene, or cell. Amutant or modified protein or enzyme is usually, although notnecessarily, expressed from a mutant polynucleotide or gene.

A “mutation” means any process or mechanism resulting in a mutantprotein, enzyme, polynucleotide, gene, or cell. This includes anymutation in which a protein, enzyme, polynucleotide, or gene sequence isaltered, and any detectable change in a cell arising from such amutation. Typically, a mutation occurs in a polynucleotide or genesequence, by point mutations, deletions, or insertions of single ormultiple nucleotide residues. A mutation includes polynucleotidealterations arising within a protein-encoding region of a gene as wellas alterations in regions outside of a protein-encoding sequence, suchas, but not limited to, regulatory or promoter sequences. A mutation ina gene can be “silent”, i.e., not reflected in an amino acid alterationupon expression, leading to a “sequence-conservative” variant of thegene. This generally arises when one amino acid corresponds to more thanone codon. Table 1 outlines which amino acids correspond to whichcodon(s). TABLE 1 Amino Acids, Corresponding Codons, andFunctionality/Property Amino Acid SLC DNA codons Side Chain PropertyIsoleucine I ATT, ATC, ATA Hydrophobic Leucine L CTT, CTC, CTA, CTG,TTA, TTG Hydrophobic Valine V GTT, GTC, GTA, GTG HydrophobicPhenylalanine F TTT, TTC Aromatic side chain Methionine M ATG Sulphurgroup Cysteine C TGT, TGC Sulphur group Alanine A GCT, GCC, GCA, GCGHydrophobic Glycine G GGT, GGC, GGA, GGG Hydrophobic Proline P CCT, CCC,CCA, CCG Secondary amine Threonine T ACT, ACC, ACA, ACG Aliphatichydroxyl Serine S TCT, TCC, TCA, TCG, AGT, AGC Aliphatic hydroxylTyrosine T TAT, TAC Aromatic side chain Tryptophan W TGG Aromatic sidechain Glutamine Q CAA, CAG Amide group Asparagine N AAT, AAC Amide groupHistidine H CAT, CAC Basic side chain Glutamic acid E GAA, GAG Acidicside chain Aspartic Acid D GAT, GAC Acidic side chain Lysine K AAA, AAGBasic side chain Arginine R CGT, CGC, CGA, CGG, AGA AGG, Stop codonsStop TAA, TAG, TGA

“Function-conservative variants” are proteins or enzymes in which agiven amino acid residue has been changed without altering overallconformation and function of the protein or enzyme, including, but notlimited to, replacement of an amino acid with one having similarproperties, including polar or non-polar character, size, shape andcharge (see Table 1).

Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and canbe, for example, at least 30%, preferably at least 50%, more preferablyat least 70%, even more preferably 80%, and most preferably at least90%, as determined according to an alignment scheme. As referred toherein, “sequence similarity” means the extent to which nucleotide orprotein sequences are related. The extent of similarity between twosequences can be based on percent sequence identity and/or conservation.“Sequence identity” herein means the extent to which two nucleotide oramino acid sequences are invariant. “Sequence alignment” means theprocess of lining up two or more sequences to achieve maximal levels ofidentity (and, in the case of amino acid sequences, conservation) forthe purpose of assessing the degree of similarity. Numerous methods foraligning sequences and assessing similarity/identity are known in theart such as, for example, the Cluster Method, wherein similarity isbased on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA(Lipman and Pearson, 1985; Pearson and Lipman, 1988). When using all ofthese programs, the preferred settings are the default settings, orthose that results in the highest sequence similarity.

As used herein, amino acid residues are “adjacent” when they are within15, preferably within 10, and more preferably within 7 Angstroms fromeach other in the 3-dimensional enzyme or protein structure. The enzymestructure can be the structure when bound to substrate or not bound tosubstrate. Adjacent amino acid residues can be next to each other in theprimary amino acid sequence or they can be adjacent as a result of thefolded structure. Preferably, no other amino acid fully or partiallyblocks direct interaction between adjacent amino acid residues.

The “activity” of an enzyme is a measure of its ability to catalyze areaction, i.e., to “function”, and may be expressed as the rate at whichthe product of the reaction is produced. For example, enzyme activitycan be represented as the amount of product produced per unit of time orper unit of enzyme (e.g., concentration or weight), or in terms ofaffinity or dissociation constants. Preferred activity units forexpressing activity include the catalytic constant (k_(cat)=V_(max)/E;V_(max) is maximal turnover rate; E is concentration of enzyme); theMichaelis-Menten constant (K_(m)); and k_(cat)/K_(m). Such units can bedetermined using well-established methods in the art of enzymes.

The “stability” or “resistance” of an enzyme means its ability tofunction, over time, in a particular environment or under particularconditions. One way to evaluate stability or resistance is to assess itsability to resist a loss of activity over time, under given conditions.Enzyme stability can also be evaluated in other ways, for example, bydetermining the relative degree to which the enzyme is in a folded orunfolded state. Thus, one enzyme has improved stability or resistanceover another enzyme when it is more resistant than the other enzyme to aloss of activity under the same conditions, is more resistant tounfolding, or is more durable by any suitable measure. For example, amore “organic-solvent” resistant enzyme is one that is more resistant toloss of structure (unfolding) or function (enzyme activity) when exposedto an organic solvent or co-solvent (e.g., DMSO, tetrahydrofuran (THF),methanol, ethanol, propanol, dioxane, or dimethylformamide (DMF)).

The “thermostability” of an enzyme means its ability to function,optionally function over time, in at elevated temperatures. One way toevaluate thermostability is to assess the ability of the enzyme toresist a loss of activity over time at various temperatures. A more“thermostable” enzyme is more resistant to at least one of loss ofstructure (unfolding) or function (enzyme activity) when exposed tohigher temperatures, for example, at temperatures of at least 35,preferably at least 45, and, even more preferably, at least 55 degreesCelsius. Thermostability can be evaluated by determining the temperature(T₅₀) at which half of the enzyme population is unfolded after a10-minute incubation. Thermostability can also be compared and expressedas the temperature at which half of the initial activity is retainedafter a 10 minute incubation after an increase from one temperature toanother, i.e., from X° C. to Y degrees ° C.

The term “substrate” means any substance or compound that is convertedor meant to be converted into another compound by the action of anenzyme catalyst. The term includes aromatic and aliphatic compounds, andincludes not only a single compound, but also combinations of compounds,such as solutions, mixtures and other materials which contain at leastone substrate. Preferred substrates for hydroxylation using thecytochrome P450 enzymes of the invention includepara-nitrophenoxycarboxylic acids (“pNCAs”) such as 12-pNCA, as well asdecanoic acid, styrene, myristic acid, lauric acid, and other fattyacids and fatty acid-derivatives. For alkane/alkene-substrates, propane,propene, ethane, ethene, butane, butene, pentane, pentene, hexane,hexene, cyclohexane, octane, octene, p-nitrophenoxyoctane (8-pnpane),and various derivatives thereof, can be used. The term “derivative”refers to the addition of one or more functional groups to a substrate,including, but not limited, alcohols, amines, halogens, thiols, amides,carboxylates, etc.

The term “cofactor” refers any substance that is necessary or beneficialto the activity of an enzyme. A “coenzyme” means a proteinaceouscofactor that interacts directly with and serves to promote a reactioncatalyzed by an enzyme. Many coenzymes also serve as carriers. Forexample, NAD+ and NADP+ carry hydrogen atoms from one enzyme to another(in the form NADH and NADPH, respectively). An “ancillary protein” meansany protein substance that is necessary or beneficial to the activity ofan enzyme.

The terms “oxygen donor”, “oxidizing agent” and “oxidant” mean asubstance, molecule or compound which donates oxygen to a substrate inan oxidation reaction. Typically, the oxygen donor is reduced (acceptselectrons). Exemplary oxygen donors, which are not limiting, includemolecular oxygen or dioxygen (O2) and peroxides, including alkylperoxides such as t-butyl hydroperoxide, cumene hydroperoxide, peraceticacid, and most preferably hydrogen peroxide (H₂O₂). A “peroxide” is anycompound other than molecular oxygen (O₂) having two oxygen atoms boundto each other.

An “oxidation enzyme” is an enzyme that catalyzes one or more oxidationreactions, typically by adding, inserting, contributing or transferringoxygen from a source or donor to a substrate. Such enzymes are alsocalled oxidoreductases or redox enzymes, and encompasses oxygenases,hydrogenases or reductases, oxidases and peroxidases. An “oxidase” is anoxidation enzyme that catalyzes a reaction in which molecular oxygen(dioxygen or O2) is reduced, for example by donating electrons to (orreceiving protons from) hydrogen.

A “luminescent” substance means any substance which produces detectableelectromagnetic radiation, or a change in electromagnetic radiation,most notably visible light, by any mechanism, including color change, UVabsorbance, fluorescence and phosphorescence. Preferably, a luminescentsubstance according to the invention produces a detectable color,fluorescence or UV absorbance. The term “chemiluminescent agent” meansany substance which enhances the detectability of a luminescent (e.g.,fluorescent) signal, for example by increasing the strength or lifetimeof the signal.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins and enzymes. These terms include double or single strandedgenomic and cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and anti-sense polynucleotide (althoughonly sense stands are being represented herein). This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNAhybrids, as well as “protein nucleic acids” (PNA) formed by conjugatingbases to an amino acid backbone. This also includes nucleic acidscontaining modified bases, for example thio-uracil, thio-guanine andfluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.).

A “coding sequence” or a sequence “encoding” a polypeptide, protein orenzyme is a nucleotide sequence that, when expressed, results in theproduction of that polypeptide, protein or enzyme, i.e., the nucleotidesequence encodes an amino acid sequence for that polypeptide, protein orenzyme. A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.Preferably, the coding sequence is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxyl) terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e.g., mammalian) DNA, and even syntheticDNA sequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription. A gene encoding a protein of theinvention for use in an expression system, whether genomic DNA or cDNA,can be isolated from any source, particularly from a human cDNA orgenomic library. Methods for obtaining genes are well known in the art,e.g., Sambrook et al (supra).

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining this invention, thepromoter sequence is bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background.

Polynucleotides are “hybridizable” to each other when at least onestrand of one polynucleotide can anneal to another polynucleotide underdefined stringency conditions. Stringency of hybridization isdetermined, e.g., by (a) the temperature at which hybridization and/orwashing is performed, and (b) the ionic strength and polarity (e.g.,formamide) of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two polynucleotides containsubstantially complementary sequences; depending on the stringency ofhybridization, however, mismatches may be tolerated. Typically,hybridization of two sequences at high stringency (such as, for example,in an aqueous solution of 0.5×SSC at 65° C.) requires that the sequencesexhibit some high degree of complementarity over their entire sequence.Conditions of intermediate stringency (such as, for example, an aqueoussolution of 2×SSC at 65° C.) and low stringency (such as, for example,an aqueous solution of 2×SSC at 55° C.), require correspondingly lessoverall complementarity between the hybridizing sequences. (1×SSC is0.15 M NaCl, 0.015 M Na citrate.) Polynucleotides that hybridize includethose which anneal under suitable stringency conditions and which encodepolypeptides or enzymes having the same function, such as the ability tocatalyze an oxidation, oxygenase, or coupling reaction of the invention.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include bacteria (e.g., E. coli and B.subtilis) or yeast (e.g., S. cerevisiae) host cells and plasmid vectors,and insect host cells and Baculovirus vectors. As used herein, a “facileexpression system” means any expression system that is foreign orheterologous to a selected polynucleotide or polypeptide, and whichemploys host cells that can be grown or maintained more advantageouslythan cells that are native or heterologous to the selectedpolynucleotide or polypeptide, or which can produce the polypeptide moreefficiently or in higher yield. For example, the use of robustprokaryotic cells to express a protein of eukaryotic origin would be afacile expression system. Preferred facile expression systems include E.coli, B. subtilis and S. cerevisiae host cells and any suitable vector.

The term “transformation” means the introduction of a foreign (i.e.,extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. The introduced gene or sequence may includeregulatory or control sequences, such as start, stop, promoter, signal,secretion, or other sequences used by the genetic machinery of the cell.A host cell that receives and expresses introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a differentgenus or species.

The terms “vector”, “vector construct” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors typically comprise the DNA of a transmissible agent,into which foreign DNA encoding a protein is inserted by restrictionenzyme technology. A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA, that canreadily accept additional (foreign) DNA and which can readily introducedinto a suitable host cell. A large number of vectors, including plasmidand fungal vectors, have been described for replication and/orexpression in a variety of eukaryotic and prokaryotic hosts.Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids,pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids(Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs,Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g., antibiotic resistance, and one or moreexpression cassettes. Preferred vectors are described in the Examples,and include without limitations pcWori+ (FIG. 3), pET-26b(+), pXTD14,pYEX-S1, pMAL, and pET22-b(+). Other vectors may be employed as desiredby one skilled in the art. Routine experimentation in biotechnology canbe used to determine which vectors are best suited for used with theinvention, if different than as described in the Examples. In general,the choice of vector depends on the size of the polynucleotide sequenceand the host cell to be employed in the methods of this invention.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Apolynucleotide or polypeptide is expressed recombinantly, for example,when it is expressed or produced in a foreign host cell under thecontrol of a foreign or native promoter, or in a native host cell underthe control of a foreign promoter.

A polynucleotide or polypeptide is “over-expressed” when it is expressedor produced in an amount or yield that is substantially higher than agiven base-line yield, e.g. a yield that occurs in nature. For example,a polypeptide is over-expressed when the yield is substantially greaterthan the normal, average or base-line yield of the nativepolypolypeptide in native host cells under given conditions, for exampleconditions suitable to the life cycle of the native host cells.

“Isolation” or “purification” of a polypeptide or enzyme refers to thederivation of the polypeptide by removing it from its originalenvironment (for example, from its natural environment if it isnaturally occurring, or form the host cell if it is produced byrecombinant DNA methods). Methods for polypeptide purification arewell-known in the art, including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix. Alternatively, antibodies producedagainst the protein or against peptides derived therefrom can be used aspurification reagents. Other purification methods are possible. Apurified polynucleotide or polypeptide may contain less than about 50%,preferably less than about 75%, and most preferably less than about 90%,of the cellular components with which it was originally associated. A“substantially pure” enzyme indicates the highest degree of purity whichcan be achieved using conventional purification techniques known in theart.

The 3-dimensional conformation of a P450 enzyme can be determined byX-ray crystallography techniques known to the skilled artisan, or may,in the case where crystallographic data is already publicly available,be simply visualized using software such as the free-ware program SwissPDB Viewer (available via the ExPASy (Expert Protein Analysis System)proteomics server of the Swiss Institute of Bioinformatics (SIB)website). For example, crystallographic data for the P450 BM-3 hemedomain has been published (Li and Poulos, 1994). The same type ofsoftware can be applied for determining the distances between selectedamino acid residues in the properly conformed wild-type enzyme, or todetermine which amino acid residues lie within a selected radius from areference residue. Such techniques are described at, e.g., the SwissPDB-viewer web site (accessed via the U.S. web site of expasy.org/spdbvon Aug. 7, 2003).

Crystal structures of wildtype P450 enzymes such as BM-3 with andwithout substrate reveal large conformational changes upon substratebinding at the active site (Haines et al., 2001; Li and Poulos, 1997;Paulsen and Ornstein, 1995; and Chang and Loew, 2000). The substratefree structure displays an open access channel with 17 to 21 orderedwater molecules. Substrate recognition serves as a conformationaltrigger to close the channel, which dehydrates the active site,increases the redox potential, and allows dioxygen to bind to the heme.

Thermostabilizing mutations may be found in amino acid residues adjacentto an amino acid residue in which an activity or peroxygenase mutationhas been introduced in the conformation where the P450 enzyme is with orwithout substrate. The skilled artisan can determinet whether thedistance between residues should be determined when the enzyme hassubstrate bound or not on a case-by-case basis. For example, this maydepend on whether the enzyme will be stored with substrate bound, orused with a particular substrate after storage. Although thermaldenaturation may occur over time regardless of whether substrate isbound, many enzymes can be stabilized by the presence of substrate.However, in most thermostability studies of P450 enzymes conducted sofar, thermal inactivation is usually measured in the absence ofsubstrate.

Suitable non-P450 BM-3 enzymes preferably have a heme domain at least30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identityto SEQ ID NO:3. In an alternative embodiment, the cDNA encoding thenon-P450 BM-3 enzymes can hybridize to cDNA encoding SEQ ID NO:3 underconditions of low, medium, or high stringency. Such hybridizationconditions are well known in the art. Preferably, although notnecessarily, the amino acid substitutions of the invention which are innon-P450 BM-3 enzymes are in conserved residues. FIGS. 4A and 4B showalignment of non-BM-3 enzymes with SEQ ID NO:3, and indicates whichresidues are identical (“*”), and conserved (“:”). For example, theresidues aligned with residue L52, F87, H100, S106, M145, A184, M237,S274, V340, and K434 in P450 BM-3 are identical or conserved.

While many P450 enzymes may not share a high sequence similarity, theheme-containing domains of P450s do display close structural similarity(Miles et al., 2000). The heme domain (P450_(BM-P)) can correspond tothe first 464 (SEQ ID NO:3) or 472 amino acid residues of a full-lengthsequence corresponding to P450 BM-3. Therefore, the positions of thevarious mutations described for the P450 BM-3 heme domain could betranslated to similar positions in different P450s having very lowsequence similarity to P450 BM-3 using molecular modeling of those P450sbased on sequence homology. Examples of using such techniques to modelvarious P450s based on sequence homology with P450 BM-3 are available(Lewis et al., 1999). The same mutations described here, when placed intheir corresponding positions in other P540 structures (as determined bymodeling) would confer similar improvements in peroxide shunt pathwayactivity and/or thermostability. In this regard, FIG. 5 shows atopological view of a cytochrome P450 enzyme, including the variousdomains, herein also termed “secondary structural elements”, ofcytochrome P450 enzymes and the mutations contemplated by the presentinvention in each of those domains. While the topological view presentedin FIG. 5 is that of P450_(BM-P), with only minor modifications, thistopology diagram may be used for other P450s.

The activity of P450 BM-3 on saturated fatty acids follows the orderC₁₅=C₁₆>C₁₄>C₁₇>C₁₃>C₁₈>C₁₂ (Oliver et al., 1997). On the C₁₆ fattyacid, k_(cat)=81 s⁻¹ and K_(m)=1.4×10⁻⁶ M (k_(cat)/K_(m)=6.0×10⁷M⁻¹s⁻¹). With the C₁₂ fatty acid, k_(cat)=26 s⁻¹, K_(m)=136×10⁻⁶ M andk_(cat)/K_(m)=1.9×10⁵ M⁻¹s⁻¹ (Oliver et al., 1997). Usually, there islittle difference in activity if the C-terminal portion of the hemedomain is truncated or substituted. For example, if the last 9-10residues are substituted for a 6-histidine-tag (“His₆”) or some othersuitable peptide sequence, or deleted, the oxidation capacity of theheme domain is not significantly affected. One of skill in the art caneasily determine whether a substitution in or deletion of one or moreamino acids in the C-terminal sequence affects the heme domain activityor thermostability.

Described herein are several mutations that have been identified toimprove the thermostability of P450 peroxygenases. Thus, a P450 variantof the invention can comprise at least one of these thermostabilizingmutations, optionally in combination with another mutations selectedfrom the ones described in Table 2A, a mutation not described in Table1A, or no other mutation. The variant P450 enzymes of the inventionretain or achieve a higher ability to use the peroxide-shunt pathway, alesser or no dependency on cofactor, and/or a higher thermostability,than the corresponding wild-type P450. Preferred amino acid mutationsare those listed in Table 2A. The skilled artisan could easily identifyother P450 variants, including variants comprising truncated, deleted,and inserted amino acid sequences, that comprise one or more of thesemutations and that show enhanced peroxide-utilization andthermostability in a suitable assay as compared to the correspondingwild-type P450.

Table 2A described preferred mutation sites for P450 variants (leftcolumn), wherein methionine is position zero. Also indicated withinparenthesis after each mutated amino acid residue is the location of theamino acid residue (compare to FIG. 5). Preferably, although notnecessarily, the amino acid substitution is among those set forth in theright column of Table 2A. A P450 BM-3 full-length or, more preferably,heme domain variants can comprise at least one, preferably at leastthree, and more preferably at least 7, and even more preferably elevenof the amino acid mutations in Table 2A. Optimally, the P450 variantincludes at least one mutation in an amino acid residue selected fromL52, S106, A184, and V340. Exemplary P450 mutants include those thathave at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:3 and comprise at least one of themutations in Table 2A.

In one embodiment, a P450 BM-3 peroxygenase variant comprises mutationsat amino acid residues F87, H100, M145, M237, S274, and/or K434. Inanother preferred embodiment, the P450 BM-3 variant also comprises amutation in one or more of L52, S106, A184, and V340. Most preferably,the mutations are L521, F87A, H100R, S106R, M145V, M145A, A184V, M237L,S274T, V340M, and K434E. Optionally, residue 469 is deleted. However,also contemplated and encompassed by the present invention are aminoacid mutations at these positions which are function-conservative to theaforementioned amino acid substitutions. For example, the mutationsM145V, M145A, M1451, and M145G, are function-conserved variants becausethe methionine has been replaced by a hydrophobic amino acid residue.TABLE 2A Cytochrome P450 Mutated Amino Acid Residues, their Location andMutations Amino Acid Residue of SEQ ID NOS: 3 (Location) Amino AcidMutation K9 (N-terminal) K9I L52 (beta sheet 1-2) L52I I58 (helix B)I58V F87 (loop between helices B′ & C - F87A or F87S lies above heme(distal side) E93 (start of helix C) E93G H100 (helix C) H100R S106(loop between helices C & D) S106R F107 (loop between helices C & D)F107L K113 (start of helix D) K113E A135 (loop between helices D & E)A135S M145 (helix E) M145V or M145A A184 (helix F) A184V N186 (helix F)N186S D217 (helix G) D217V M237 (helix H) M237L N239 (end of helix H)N239H E244 (loop between helix H and E244G beta sheet 5-1) S274 (helixI) S274T L324 (end of helix K) L324I V340 (beta sheet 2-1) V340M I366(helix K″) I366V K434 (beta sheet 4-1) K434E E442 (end of beta sheet4-2) E442K V446 (beta sheet 3-2) V446I H469 (His-tag) deleted

In addition, the invention provides P450 BM-3 mutants having specificnucleic acid and amino acid sequences. The nucleic acid sequencesinclude those which encode the P450 BM-3 variants represented in Table2B, where the right column lists the amino acid mutations present ineach specific variant enzyme. The amino acid sequences include thosewhich have the combinations of amino acid mutations in Table 2B, whereall mutations refer to SEQ ID NOS:2 or 3, starting at position zero. Thepresent invention also provides P450 BM-3 nucleic acids encoding silentmutations, as described in the Examples. A particularly preferred mutantaccording to the present invention is 5H6. TABLE 2B P450 BM-3Full-Length or Heme Domain Peroxygenases Amino Acid Mutations inWild-Type P450 BM-3 (SEQ ID NO: 2) or Wild-Type P450 BM-3 DesignationHeme Domain (SEQ ID NO: 3) 2H1 K434E 1F8 K9I, H100R 2E10 K113E, K434E2E10-1 F87A, K113E, D217V, and K434E 2E10-3 F87A, E93G, K113E, N186S,and K434E 2E10-4 F87A, K113E, M237L, and K434E step B3 F87A, H100R,M145V, S274T, and K434E step B6 F87A, H100R, M145V, M237L, and K434E21B3 I58V, F87A, H100R, F107L, A135S, M145V, N239H, S274T, K434E, andV446I TH3 I58V, F87A, H100R, F107L, A135S, M145V, N239H, S274T, L324I,I366V, K434E, E442K, and V446I TH4 I58V, F87A, H100R, F107L, A135S,M145A, N239H, S274T, L324I, I366V, K434E, E442K, and V446I 5H6 L52I,I58V, F87A, H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I,V340M, I366V, K434E, E442K, V446I, and deletion of H469.

A peroxygenase mutant has a peroxide-driven oxidation activity at leasttwice, more preferably at least five, and even more preferably at least100 times that of the corresponding wild-type P450 in the absence ofco-factor, can be thermostabilized as described herein. Preferably, theperoxygenase is a variant of a P450 BM-3 heme domain. The P450 BM-3variants of the invention have an at least two-fold improvement in theability to oxidize a chosen substrate in the absence of co-factor andpresence of H₂O₂ as compared to either wild-type P450 BM-3 or the F87Amutant, or the heme domains thereof. Even more preferably, theimprovement for this property as compared to wild-type is at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 40-fold, or at least 80-fold. For peroxide activitycompared to F87A, the improvements for this property is at least 10-foldto about 20-fold. The peroxide-driven oxidation activity of the P450BM-3 variant can, in addition, be at least 10 times that of the mutantF87A.

As shown in the examples, F87A in combination with H100R, M145A, M145V,M237L, S274T, and K434E were noted as especially effective mutations forimproving peroxide-shunt activity. These mutations were present inproducts of recombination, in which the point mutations of severaldifferent mutants, (each with different point mutations accumulated fromseveral rounds of error-prone PCR), were allowed to assemble in allcombinations. In this manner, improved recombinant products with onlybeneficial or neutral mutations can be screened for and isolated, andall deleterious mutations removed. Mutation K434E was also noted to haveappeared in two separately evolved mutants (“2H1” and “2E10”), againindicating that this mutation is especially effective in improvingperoxide shunt activity. It was also found that F87S supported the shuntpathway better than wild-type, although to a lesser degree than F87A.

The peroxygenase variant may comprise a first mutation at a positioncorresponding to F87 of SEQ ID NO:3 and at least one second mutation ina secondary structure element of the heme domain selected from the groupconsisting of the N-terminus, β1-2, helix B, a loop between helices B′and C, helix C, a loop between helices C and D, helix D, a loop betweenhelices D and E, helix E, helix F, helix G, helix H, a loop betweenhelix H and beta sheet (β) 5-1, helix I, helix K, helix K″, β4-1, β4-2,and β3-2. The at least one second mutation can be in a secondarystructural element selected from the group consisting of the loopbetween helices B′ and C, helix C, helix I, and β4-1, or may be acombination thereof. In a preferred embodiment, the isolated nucleicacid encodes a variant having a higher thermostability than the parent.For example, the mutation in the loop between helices B′ and C is at anamino acid residue corresponding to amino acid residue F87 of SEQ IDNO:3, the mutation in β1-2 is at an amino acid residue corresponding toamino acid residue L52 of SEQ ID NO:3, the mutation in helix C is at anamino acid residue corresponding to an amino acid residue of SEQ ID NO:3selected from E93 and H100, the mutation in the loop between helices Cand D is at an amino acid residue corresponding to an amino acid residueof SEQ ID NO:3 selected from S106 and F107, the mutation in helix E isat an amino acid residue corresponding to amino acid residue M145 of SEQID NO:3, the mutation in helix F is at an amino acid residuecorresponding to an amino acid residue of SEQ ID NO:3 selected from A184and N186, the mutation in helix H is at an amino acid residuecorresponding to an amino acid residue of SEQ ID NO:3 selected from M237and N239, the mutation in helix I is at an amino acid residuecorresponding to amino acid residue S274 of SEQ ID NO:3, the mutation inhelix K is at an amino acid residue corresponding to an amino acidresidue of SEQ ID NO:3 selected from L324 and V340, and the mutation inβ4-1 is at an amino acid residue corresponding to amino acid residueK434 of SEQ ID NO:3.

These peroxygenases can then be modified to increase thermostability ascompared to the peroxygenase variant, preferably to the same level asthe wild-type P450, and even more preferably to a higher thermostabilitythan the wild-type P450. In this thermostabilization process, theperoxygenase capability remains higher than that of the wild-type P450.As shown in Examples 4 and 5, mutations suitable for improvingthermostability, preferably while retaining or improving oxidationactivity via peroxide shunt pathway, include L521, S106R, M145A, A184V,L324I, V340M, 1366V, and E442K. In one embodiment, the thermostabilizingmutations are located in proximity to a mutation which improvesoxygenase activity via the peroxide shunt pathway. For example, thethermostabilizing mutation may be located in an adjacent secondarystructural element or no more than about 50, preferably no more than 20,and even more preferably no more than 10 amino acids from a mutationimproving activity. In a particular embodiment, the thermostabilizingmutations stabilize a P450 BM-3 mutant comprising at least one,preferably at least two, and even more preferably all of the mutations158V, F107L, S274T, and K434E. Accordingly, a P450 BM-3 variantcomprising at least one, preferably at least two, and most preferablyall of these mutations, or a nucleic acid encoding such mutants, is apreferred embodiment of the invention. In addition, amino acids whichare function-conservative to the amino acid introduced instead of thewild-type amino acid can be used as well. For example, at residue M145,the methionine can be substituted for an alanine, valine, isoleucine,glycine, or any other hydrophobic amino acid (see Table 3) to create avariant P450 BM-3 of the invention.

Moreover, peroxygenase variants may be derived from P450 enzymes otherthan P450 BM-3. These peroxygenases have a higher ability to useperoxide as an oxygen donor, and a lesser or no dependency on cofactor.In particular, one may construct a P450 peroxygenase mutant based on thesequence of a non-P450 BM-3 enzyme by aligning the sequences andidentifying those residues in the non-P450 BM-3 sequence that correspondto the following residues of SEQ ID NO:2: K9, 158, F87, E93, H100, F107,K113, A135, M145, M145, N186, D217, M237, N239, E244, S274, L324, 1366,K434, E442, and V446. Once one has identified the residues of thenon-P450 BM-3 enzyme that correspond to those of identified above fromSEQ ID NOS:2 or 3, one may make an appropriate amino acid substitutionto derive a peroxygenase variant. For example, CYP102A3 or CYPE BACSU(GenBank Accession No. 008336) is a P450 that can be used to make avariant of the present invention. The heme domain of CYP102A3 has 67%identity to that of P450 BM-3. By aligning the heme domains of CYP102A3and P450 BM-3, one can identify those residues of CYP102A3 thatcorrespond with the P450 BM-3 residues identified in Table 2A and makelike substitutions to the CYP102A3 sequence. Another example is theK434E mutation, which could be translated into a K437E mutation in theP450 enzyme GenBank Accession No. A69975. These and other exemplarynon-BM-3 enzymes are identified in Table 3, but the skilled artisancould identify other P450s that may be modified in accordance with thepresent invention. TABLE 3 Preferred Non-BM3 Variants % Identity of HemeDomain GenBank Non-BM-3 to P450 BM-3 Accession Number enzyme OrganismHeme Domain (SEQ ID NO) CYP 102A3/ Bacillus 67% O08336 (SEQ ID NO: 4)CYPE BACSU subtilis A69975 (SEQ ID NO: 5) CYP 102A2 Bacillus 66% O08394(SEQ ID NO: 6) CYPD BACSU subtilis D69799 (SEQ ID NO: 7) — Streptomyces45% CAB66201 coelicolor (SEQ ID NO: 8) A3(2) P450_(foxy) Fusarium 41%BAA82526 oxysporum (SEQ ID NO: 9) — Gibberella 36% AAG27132 moniliformis(SEQ ID NO: 10)

Any method can be used to “translate” the P450 BM-3 mutation ontoanother cytochrome P450 enzyme, and such methods are well known in theart. For example, sequence alignment software such as SIM (alignment oftwo protein sequences), LALIGN (finds multiple matching subsegments intwo sequences), Dotlet (a Java applet for sequence comparisons using thedot matrix method); CLUSTALW (available via the World Wide Web asfreeware), ALIGN (at Genestream (IGH)), DIALIGN (multiple sequencealignment based on segment-to-segment comparison, at University ofBielefeld, Germany), Match-Box (at University of Namur, Belgium), MSA(at Washington University), Multalin (at INRA or at PBIL), MUSCA(multiple sequence alignment using pattern discovery, at IBM), and AMAS(Analyse Multiply Aligned Sequences). A person of skill can choosesuitable settings, or simply use standard default settings, in theseprograms to align P450 BM-3 with another cytochrome P450 enzyme. SeeFIG. 4 for representative sequence alignments, and Table 3 forrepresentative non-BM-3 enzymes to which the mutations of the inventioncan be translated.

Alternatively, sequence alignments of P450 BM-3 with other cytochromeP450 enzymes can be taken from the literature, and amino acid residuescorresponding to the mutated amino acid residues of the inventionidentified. For example, such information can be derived from Ortiz deMontellano (1995) (see, especially, FIG. 11 on page 163 and FIG. 1 onpage 187), hereby incorporated by reference. Once the correspondingamino acid residues have been identified, a person of skill can testvarious mutations of these amino acid residues to identify those thatyield improved peroxide shunt utilization ability or improvedthermostability as compared to the cytochrome P450 wild-type enzyme.Preferably, the amino acid substitution corresponds to the one(s) listedin Table 2A for the P450 BM-3 mutation, or a function-conservative aminoacid thereof.

The non-P450 BM-3 peroxygenase variant can thereafter bethermostabilized the in accordance with the present invention. Forexample, one may identify those amino acid residues that correspond toL52, S106, M145, and/or E442 of P450 BM-3, and make a substitution inone or more of these residues. Alternatively, one may select amino acidresidues that are within 15, 10, or 7 Angstroms of one or more aminoacid residues which has been mutated to improve peroxygenase activity,create a library of variants having mutations in these residues, andscreen for improved thermostability. The mutation in the non-BM-3sequence introduced to improve peroxygenase activity preferably resultsin one or more of the following amino acid substitutions: K91, I58V,F87A, E93G, H100R, F107L, K113E, A135S, M145V, N186S, D217V, M237L,N239H, E244G, S274T, L324I, I366V, K434E, and V446I, where the aminoacid residue number refers to the corresponding P450 BM-3 residue.Similarly, the mutation in the non-BM-3 sequence introduced to improvethermostability preferably results in one or more of the following aminoacid substitutions: L52I, S106R, M145A, A184V, E442K, and V340M.

Preparation of Mutant or Variant P450 Enzymes

One technique to create peroxygenase mutants or thermostable variants ofwild-type or parent cytochrome P450 enzymes, including P450 BM-3, isdirected evolution. General methods for generating libraries andisolating and identifying improved proteins according to the inventionusing directed evolution are described briefly below. More extensivedescriptions can be found in, for example, Arnold (1998); U.S. Pat. Nos.5,741,691; 5,811,238; 5,605,793 and 5,830,721; and InternationalApplications WO 98/42832, WO 95/22625, WO 97/20078, WO 95/41653 and WO98/27230. The basic steps in directed evolution are (1) the generationof mutant libraries of polynucleotides from a parent or wild-typesequence; (2) (optional) expression of the mutant polynucleotides tocreate a mutant polypeptide library; (3) screening the polynucleotide orpolypeptide library for a desired property of a polynucleotide orpolypeptide; and (4) selecting mutants which possess a higher level ofthe desired property; and (5) repeating steps (1) to (5) using theselected mutant(s) as parent(s) until one or more mutants displaying asufficient level of the desired activity have been obtained. Theproperty can be, but is not limited to, ability to use peroxide as anoxygen source.

The parent protein or enzyme to be evolved can be a wild-type protein orenzyme, or a variant or mutant which has an improved property such asimproved peroxygenase activity or thermostability. The parentpolynucleotide can be retrieved from any suitable commercial ornon-commercial source. The parent polynucleotide can correspond to afull-length gene or a partial gene, and may be of various lengths.Preferably, the parent polynucleotide is from 50 to 50,000 base pairs.It is contemplated that entire vectors containing the nucleic acidencoding the parent protein of interest may be used in the methods ofthis invention.

Whether applied in the contaxt of directed evolution or specific proteindesign based on modelling, any method can be used for generatingmutations in the parent polynucleotide sequence to provide a library ofevolved polynucleotides, including error-prone polymerase chainreaction, cassette mutagenesis (in which the specific region optimizedis replaced with a synthetically mutagenized oligonucleotide),oligonucleotide-directed mutagenesis, parallel PCR (which uses a largenumber of different PCR reactions that occur in parallel in the samevessel, such that the product of one reaction primes the product ofanother reaction), random mutagenesis (e.g., by random fragmentation andreassembly of the fragments by mutual priming); site-specific mutations(introduced into long sequences by random fragmentation of the templatefollowed by reassembly of the fragments in the presence of mutagenicoligonucleotides); parallel PCR (e.g., recombination on a pool of DNAsequences); sexual PCR; and chemical mutagenesis (e.g., by sodiumbisulfite, nitrous acid, hydroxylamine, hydrazine, formic acid, or byadding nitrosoguanidine, 5-bromouracil, 2-aminopurine, and acridine tothe PCR reaction in place of the nucleotide precursor; or by addingintercalating agents such as proflavine, acriflavine, quinacrine);irradiation (X-rays or ultraviolet light, and/or subjecting thepolynucleotide to propagation in a host cell that is deficient in normalDNA damage repair function); or DNA shuffling (e.g., in vitro or in vivohomologous recombination of pools of nucleic acid fragments orpolynucleotides). Any one of these techniques can also be employed underlow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence, or to mutagenize a mixture offragments of unknown sequence. The following sections describe some ofthe mutagenesis techniques that can be employed to generate the productsof the invention.

Error prone PCR is a well-known technique relying on, for example, theintrinsic infidelity of Taq-based PCR, which can be used to mutate ormutagenize a mixture of fragments of unknown sequences (Caldwell, R. C.;Joyce, G. F. PCR Methods Applic. 2, 28 (1992); Leung, D. W. et al.Technique 1, (1989); Gramm, H. et al. Proc. Natl. Acad. Sci. USA 89,3576 (1992)).

Cassette mutagenesis (Stemmer, W. P. C. et al. Biotechniques 14, 256(1992); Arkin, A. and Youvan, D.C. Proc. Natl. Acad. Sci. USA 89, 7811(1992); Oliphant, A. R. et al. Gene 44, 177 (1986); Hermes, J. D. et al.Proc. Natl. Acad. Sci. USA 87, 696 (1990); Delagrave et al. ProteinEngineering 6, 327 (1993); Delagrave et al. Bio/Technology 11, 1548(1993); Goldman, E. R. and Youvan D.C. Bio/Technology 10,1557 (1992)),is a technique in which the specific region optimized is replaced with asynthetically mutagenized oligonucleotide. These techniques can also beemployed under low fidelity polymerization conditions to introduce a lowlevel of point mutations randomly over a long sequence, or to mutagenizea mixture of fragments of unknown sequence.

Oligonucleotide directed mutagenesis, which replaces a short sequencewith a synthetically mutagenized oligonucleotide, may also be employedto generate evolved polynucleotides having improved expression or novelsubstrate specificity.

Alternatively, nucleic acid shuffling, which uses a method of in vitroor in vivo, generally homologous, recombination of pools of nucleic acidfragments or polynucleotides, can be employed to generate polynucleotidemolecules having variant sequences of the invention.

The polynucleotide sequences for use in the invention can also bealtered by chemical mutagenesis. Chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other agents that are analogues of nucleotide precursors includenitrosoguanidine, 5 bromouracil, 2 aminopurine, or acridine. Generally,these agents are added to the PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.Random mutagenesis of the polynucleotide sequence can also be achievedby irradiation with X rays or ultraviolet light, or by subjecting thepolynucleotide to propagation in a host (such as E. coli) that isdeficient in the normal DNA damage repair function. Generally, plasmidDNA or DNA fragments so mutagenized are introduced into E. coli andpropagated as a pool or library of mutant plasmids.

Where there are regions of known or suspected importance for an enzymeactivity or property, saturation mutagenesis has proven useful togenerate mutants with improved functions. In this technique,particularly suitable for preparing a library of mutations in an aminoacid close to an amino acid mutated to introduce peroxygenase activity,a pool of mutants with all possible amino acid substitutions at one ormore residues of interest is generated, and mutants with desiredproperties are isolated by an efficient selection or screening procedure(Miyazaki, K. and Arnold, F. H. (1999) J. Mol. Evol. 49, 716-720.Howitz, M. S. and Loeb, L. A. (1986). Proc. Natl. Acad. Sci. USA. 83,7406-7409). Commercially available kits, such as the QuikChange7Site-Directed Mutagenesis kit (Stratagene) can be used to carry outsaturation mutagenesis. The QuikChange7 kit allows for point mutationsto be made without performing error-prone PCR, thus allowing for a highdegree of accuracy. A “saturation mutagenesis library” is a library ofvariants of a parent protein, wherein each variant protein has amutation in the same amino acid residue.

Once the evolved polynucleotide molecules are generated they can becloned into a suitable vector selected by the skilled artisan accordingto methods well known in the art. If a mixed population of the specificnucleic acid sequence is cloned into a vector it can be clonallyamplified by inserting each vector into a host cell and allowing thehost cell to amplify the vector and/or express the mutant or variantprotein or enzyme sequence. Any one of the well-known procedures forinserting expression vectors into a cell for expression of a givenpeptide or protein may be used. Suitable vectors include plasmids andviruses, particularly those known to be compatible with host cells thatexpress oxidation enzymes or oxygenases. E. coli is one exemplarypreferred host cell. Other exemplary cells include other bacterial cellssuch as Bacillus and Pseudomonas, archaebacteria, yeast cells such asSaccharomyces cerevisiae, insect cells and filamentous fungi such as anyspecies of Aspergillus cells. For some applications, plant, human,mammalian or other animal cells may be preferred. Suitable host cellsmay be transformed, transfected or infected as appropriate by anysuitable method including electroporation, CaCl₂ mediated DNA uptake,fungal infection, microinjection, microprojectile transformation, viralinfection, or other established methods.

The mixed population of polynucleotides or proteins may then be testedor screened to identify the recombinant polynucleotide or protein havinga higher level of the desired activity or property. Themutation/screening steps can then be repeated until the selectedmutant(s) display a sufficient level of the desired activity orproperty. Briefly, after the sufficient level has been achieved, eachselected protein or enzyme can be readily isolated and purified from theexpression system, or media, if secreted. It can then be subjected toassays designed to further test functional activity of the particularprotein or enzyme. Such experiments for various proteins are well knownin the art, and are described below and in the Examples below.

The directed evolution process can be aimed at producing enzymevariants, most preferably enzyme comprising only the entire or partialheme domain, which can use a peroxide, for example peracetic acid,t-butyl hydroperoxide, cumene hydroperoxide, or hydrogen peroxide,and/or which aremore thermostable than its parent. Mutations thatenhance the efficiency of peroxide-based oxidation by BM-3 or othercytochrome P450 enzymes can serve to enhance the peroxide shunt activityof the enzyme variants. The mutations described here can be combinedwith mutations for improving alkane-oxidation activity or organicsolvent resistance, for example, and tested for their contributions toperoxide-driven alkane and alkene oxidation.

The evolved enzymes can be used in biocatalytic processes for, e.g.,hydroxylation in the absence of molecular oxygen and cofactor, alkanehydroxylation, or for improving yield of reactions involving oxidationof substrates with low solubility in aqueous solutions. The enzymevariants of the invention can be used in biocatalytic processes forproduction of chemicals from hydrocarbons, particularly alkanes andalkenes, in soluble or immobilized form. Furthermore, the enzymevariants can be used in live cells or in dead cells, or it can bepartially purified from the cells. One preferred process would be to usethe enzyme variants in any of these forms (except live cells) in anorganic solvent, in liquid or even gas phase, or for example in asuper-critical fluid like CO₂. Another preferred process is to use theenzyme variants in laundry detergents.

The method of screening for selection of mutants or variants, forfurther testing or for the next round of mutation, will depend on thedesired property sought. For example, in this invention, polypeptidesencoded by recombinant nucleic acids which encode cytochrome P450enzymes can be screened for improved use of the “peroxide-shunt”pathway, with less or no dependency on co-factor, and/or for improvedthermostability. Such tests are well known in the art. Examplary testsare provided in the Examples.

In a broad aspect, a screening method to detect oxidation comprisescombining, in any order, substrate, oxygen donor, and test oxidationenzyme. The assay components can be placed in or on any suitable medium,carrier or support, and are combined under predetermined conditions. Theconditions are chosen to facilitate, suit, promote, investigate or testthe oxidation of the substrate by the oxygen donor in the presence ofthe test enzyme, and may be modified during the assay. The amount ofoxidation product, i.e., oxidized substrate, is thereafter detectedusing a suitable method. Further, as described in WO 99/60096, ascreening method can comprise a coupling enzyme such as horseradishperoxidase to enable or enhance the detection of successful oxidation.

In one embodiment, it is not necessary to recover test enzyme from hostcells that express them, because the host cells are used in thescreening method, in a so-called “whole cell” assay. In this embodiment,substrate, oxygen donor, and other components of the screening assay,are supplied to the transformed host cells or to the growth media orsupport for the cells. In one form of this approach, the test enzyme isexpressed and retained inside the host cell, and the substrate, oxygendonor, and other components are added to the solution or platecontaining the cells and cross the cell membrane and enter the cell.Alternatively, the host cells can be lysed so that all intracellularcomponents, including any recombinantly expressed intracellular enzymevariant, can be in direct contact with any added substrate, oxygendonor, and other components. A particularly suitable whole-cellscreening assay for P450 BM-3 mutants has been presented by Schwaneberget al. (2001).

Resulting oxygenated products are detected by suitable means. Forexample, an oxidation product may be a colored, luminescent, orfluorescent compound, so that transformed host cells that produce moreactive oxidation enzymes “light up” in the assay and can be readilyidentified, and can be distinguished or separated from cells which donot “light up” as much and which produce inactive enzymes, less activeenzymes, or no enzymes. A fluorescent reaction product can be achieved,for example, by using a coupling enzyme, such as laccase or horseradishperoxidase, which forms fluorescent polymers from the oxidation product.A chemiluminescent agent, such as luminol, can also be used to enhancethe detectability of the luminescent reaction product, such as thefluorescent polymers. Detectable reaction products also include colorchanges, such as colored materials that absorb measurable visible or UVlight.

To screen for improved use of the peroxide-shunt pathway and/or a lesserdependency on NADPH co-factor for P450 BM-3 variants, a substrate suchas 12-pNCA can be added to the enzyme, and 12-pNCA conversion initiatedby adding peroxide (e.g., 1 mM H₂O₂). The rate of oxidation of the12-pNCA substrate can be monitored by measuring the change in absorbanceat 398 nm with time, which indicates the rate of formation of theco-product para-nitrophenolate (pNP).

A rapid, reproducible screen that is sensitive to small changes(<2-fold) in activity is desirable (Arnold, 1998). For example, if analkane-substrate is desired, an alkane analog such as 8-pnpane (seeExample 1), can be prepared that generates yellow color uponhydroxylation. This “surrogate” substrate with a C8 backbone and ap-nitrophenyl moiety is an analog of octane, and allows use of acolorimetric assay to conveniently screen large numbers of P450 BM-3 orother cytochrome P450 mutants for increased hydroxylation activity inmicrotiter plates (Schwaneberg et al., 1999(a); Schwaneberg et al.,2001). Hydroxylation of 8-pnpane generates an unstable hemiacetal whichdissociates to form (yellow) p-nitrophenolate and the correspondingaldehyde. The hydroxylation kinetics of hundreds of mutants can then bemonitored simultaneously in the wells of a microtiter plate using aplate reader (Schwaneberg et al., 2001). This method is particularlysuitable for detecting P450 variants with improved alkane-oxidationactivity.

Enzyme variants displaying improved levels of the desired activity orproperty in the screening assay(s) can then be expressed in higheramounts, retrieved, optionally purified, and further tested for theactivity or property of interest.

The cytochrome P450 variants can be selected for a desired property oractivity can be further evaluated by any suitable test or tests known inthe art to be useful to assess the property or activity. For example,the enzyme variants can be evaluated for their ability to use hydrogenperoxide or another peroxide as an oxygen source, their ability tofunction in the absence of co-factor, and/or their thermostability.Preferably, the activity of the corresponding wild-type P450 enzyme or a“control” variant is analyzed in parallel, as a control.

An assay for ability to use hydrogen peroxide as oxygen source and/orability to function in the absence of co-factor essentially comprisescontacting the cytochrome P450 variant with a specific amount of asubstrate such as, e.g., 12-pNCA or laurate, in the presence ofperoxide, e.g., hydrogen peroxide (H₂O₂) with low or no amounts ofoxygen donor and/or cofactor, while including any other components thatare necessary or desirable to include in the reaction mixture, such asbuffering agents. After a sufficient incubation time, the amount ofoxidation product formed, or, alternatively, the amount of intactnon-oxidized substrate remaining, is estimated. For example, the amountof oxidation product and/or substrate could be evaluatedchromatographically, e.g., by mass spectroscopy (MS) coupled tohigh-pressure liquid chromatography (HPLC) or gas chromatography (GC)columns, or spectrophotometrically, by measuring the absorbance ofeither compound at a suitable wavelength. By varying specific parametersin such assays, the Michaelis-Menten constant (K_(m)) and/or maximumcatalytic rate (V_(max)) can be derived for each substrate as is wellknown in the art. In addition, in particular by HPLC and GC techniques,particularly when coupled to MS, can be used to determine not only theamount of oxidized product, but also the identity of the product andtherefore the selectivity of the variants. For example, laurate can beoxidized at various carbon positions. When using a fatty acid surrogatesubstrate such as 12-pNCA, the kinetics of a P450 enzyme reaction can beestimated by monitoring the formation of the chromophore co-product pNPusing a spectrophotometer. The total amount of pNP formed is also easilymeasured and is a good indication of the total amount of substrateoxidized in the reaction. Peroxygenase activities can be measured atroom temperature, using a colorimetric assay with12-p-nitrophenoxycarboxylic acid (12-pNCA) as substrate. In Example 5,using such an assay, it was found that 5H6 retains ˜50% of the highactivity of 21B3 and is almost ten times as active as HF87A (Table 9).

To characterize the thermostability of a peroxygenase variant, thefraction of folded heme domain remaining after heat-treatment can bemeasured. This can be determined from the fraction of the ferrousheme-CO complex that retains the 450 nm absorbance peak characteristicof properly-folded P450. FIG. 8 shows the percentage of properly-foldedheme domain protein remaining after 10-minute incubations at different,elevated temperatures. To allow comparison to the wildtype full-lengthenzyme (BWT), whose stability is limited by the stability of thereductase domain and therefore cannot be determined from the CO-bindingmeasurement, one can determine the residual (NADPH-driven) activity ofBWT following 10-minute incubations at the same temperatures. By fittingthe data in FIG. 8 to a two-state model, half-denaturation temperaturesfor the 10-minute heat incubations (T₅₀) can be calculated. The T₅₀value thus corresponds to the temperature at which half of the enzymepopulation is denatured after 10 minutes of incubation. According to theinvention, a thermostabilized peroxygenase preferably has a T₅₀temperature higher than that of at least one of the correspondingwild-type enzyme, wild-type heme domain, or non-stabilized peroxygenaseparent. In a preferred embodiment, the T₅₀ of the thermostabilizedperoxygenase is at least 3° C., more preferably at least 5° C., evenmore preferably at least 10° C., and optimally at least 15° C. higherthan that of at least one of the corresponding wild-type enzyme,wild-type heme domain, or non-stabilized peroxygenase parent.

Another useful indicator of thermostability is to conduct an oxidationreaction at one or more temperatures. The temperatures can be in therange of, e.g., about room temperature to about 100 degrees Celsius,more preferably from about 35 degrees to about 70 degrees Celsius.Alternatively, thermostability can be evaluated by measuring the amountof room temperature activity retained following incubation at anelevated temperature. A variant's activity is measured at roomtemperature as the amount of oxidation product or bi-product formed, orremaining amount of substrate. A sample of the variant is then subjectto partial heat inactivation by incubating the sample at a controlled,elevated temperature for a set time. The sample is then rapidly cooledto room temperature and the activity of the sample is measured exactlyas the activity was measured before the inactivation. The fraction ofinitial activity retained by the incubated sample is an indicator of thethermostability of the enzyme variant, and, optionally, compared towild-type enzyme or a control variant. Such assays can be conducted atseveral temperatures and for various lengths of time.

Another useful indicator of enzyme stability comes from the rate ofinactivation at high temperature. FIG. 9 shows the percentage ofactivity that remains for different P450 enzyme variants upon heating at57.5° C. The activities decay exponentially with time (first-order), andthe half-life (t½) of each corresponding catalytic system is shown inTable 8. The heme domain of F87A (HF87A; which is less thermostable thanthe heme domain of wild-type P340 BM-3 (HWT); see FIG. 8) issignificantly more resistant to inactivation at 57.5° C. compared tofull-length wild-type P450 BM-3 (BWT). The half-life of HF87A is alsohigher than that of BWT at room temperature. The half-life of 5H6 at57.5° C. is 50 times longer than that of HF87A and 250 times that ofBWT. The fraction of peroxygenase activity remaining after heattreatment correlated with the fraction of remaining CO-binding peak forHF87A and 5H6. Residual activity of HWT cannot be correlated to theremaining CO-binding peak because HWT has essentially no peroxygenaseactivity.

EXAMPLES

The invention is illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

Example 1 Cytochrome P450 BM-3 Heme Domain Mutants More Active inPeroxide-Driven Hydroxylation

This example demonstrates the improved activity of P450 BM-3 mutantsusing hydrogen peroxide instead of NADPH.

Materials and Methods

All chemical reagents were procured from Aldrich, Sigma, or Fluka.Enzymes used for DNA manipulations were purchased from New EnglandBiolabs, Stratagene, and Boehringer Mannheim, unless otherwise noted.

All P450 enzymes described here were expressed in catalase-deficient E.coli (Nakagawa et al., 1996) using theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible pCWori+ vector(Barnes et al., 1991), which is under the control of the double Ptacpromoter and contains an ampicillin resistance coding region. Expressionwas accomplished by growth in terrific broth (TB) supplemented with 0.5mM thiamine, trace elements (Joo et al., 1999), 1 mM 6-aminolevulinicacid, and 0.5-1 mM IPTG at 30° C. for ˜18 hrs.

Library Generation

With the exception of one generation, in which the mutant library wascreated by recombination, libraries were generated under standarderror-prone PCR conditions (Zhao et al., 1999). Specifically, 100 μlreactions contained 7 mM Mg²⁺, 0.2 mM dNTPs plus excess concentrationsof dCTP and either dTTP or dATP (0.8 mM each), 20 fmole template DNA (asplasmid), 30 pmole of each outside primer, 10 μl Taq buffer (Roche) and1 μl (5 units) Taq polymerase (Roche). Due to the high concentration ofMg²⁺ and excess of two dNTPs it was determined that no Mn²⁺ wasnecessary to generate mutant libraries with a suitable fitness landscape(30% to 40% “dead” clones). PCR was performed in a PTC200 thermocycler(MJ Research). The temperature cycle used was: 94° C. for 1 min followedby 29 cycles of 94° C. for 1 min then 55° C. for 1 min then 72° C. for1:40.

One round of recombination was performed, which resulted in mutants“step B6” and “step B3”. StEP recombination was performed essentially asdescribed (Zhao et al., 1999) using HotStarTaq DNA Polymerase (Qiagen).The parent genes used for the recombination included variants “2H1”,“1F8-1”, “1F8-2”, “2E10-1”, “2E10-2”, “2E10-3”, and “2E10-4”. A 50 μlPCR reaction contained 160 ng total template DNA (comprised ofapproximately equal concentrations of the seven mutant genes), 0.2 mMdNTPs, 5 pmole outside primers, 5 μl Qiagen Hotstar buffer (containing15 mM Mg²⁺), and 2.5 U HotstarTaq polymerase. PCR was performed in aPTC200 thermocycler (MJ Research). The temperature protocol was asfollows: (hot start) 95° C. for 3 min, followed by 100 cycles of 94° C.for 30 sec and 58° C. for 8 sec.

The library that generated thermostable mutant TH4 was made using theGeneMorph PCR Mutagenesis Kit (Stratagene). A parent DNA templateconcentration of ˜500 pg/50 μl was chosen based on the resultinglibrary's suitable fitness landscape (approximately 50% of the librarycontaining essentially inactive variants).

For all PCR manipulations on the entire BM-3 heme domain gene theforward primer sequence was: 5′-ACAGGATCCATCGATGCTTAGGAGGTCATAT (SEQ IDNO:11) G-3′

and the reverse primer sequence was: 5′-GCTCATGTTTGACAGCTTATCATCG-3′.(SEQ ID NO:12)

The heme domain gene was cloned into the pCWori vector using the uniquerestriction sites BamHI at the start of the gene and EcoRI at the end.The resulting plasmid was transformed into the catalase-deficient E.coli strain and colonies were selected on agar plates containingampicillin (100 μg/ml).

Preparation of 12-pNCA

The 12-pNCA surrogate substrate was prepared as previously described(Schwaneberg et al., 1999(a)) except hydrolysis of the ester was carriedout nonenzymatically by refluxing the ester in a 1:1 mixture of THF anda basic (1 M KOH) aqueous solution. TLC and proton NMR analyses showedno detectable impurities in the isolated substrate.

P450 Quantification by CO-Binding

P450 enzyme concentrations were quantified by CO-binding differencespectra of the reduced heme as described (Omura et al., 1964). Ingeneral, 50 μl of purified enzyme or enzyme lysate was added to 750 μLof a freshly prepared solution of sodium hydrosulfite (˜10 mg/ml) andthe P450 was allowed to be reduced for about one minute. The absorbanceof this solution was then blanked in a spectrometer before bubbling COthrough the reduced enzyme solution for one minute. After another 30seconds the difference spectrum was measured from 500 nm to 400 nm, andthe absorbance value at 490 nm was subtracted from the 450 nm peak. Theextinction coefficient for all P450 enzymes was taken to be 91,000 M-1cm⁻¹ (Omura et al., 1964).

Screening for Peroxide Shunt Pathway Activity

Colonies resulting from transformation of a mutant library made byeither error-prine PCR or StEP recombination were picked into 1 mldeep-well plates containing LB media (300 μl) and ampicillin (100μg/ml). Plates were incubated at 30° C., 270 rpm, and 80% relativehumidity. After 24 hours, 20 μl of culture liquid from each well wasused to inoculate 300 μl of TB media containing ampicillin (100 μg/ml),thiamine (0.5 mM), and trace elements (Joo et al., 1999) contained in anew 1 ml deep-well plate. This plate with TB cultures was grown at 30°C., 270 rpm for approximately three hours before the cells in each wellwere induced by the addition of δ-aminolevulinic acid (1 mM) andisopropyl-β-D-thiogalactopyranoside (IPTG) (0.5 mM). Cultures were thengrown for an additional 18 hours for maximum enzyme expression. Alldeep-well plates were grown in a Kühner ISF-1-W shaker with humiditycontrol.

After cell growth the plates were centrifuged and supernatants werediscarded. Cell pellets were frozen at −20° C. before lysing. Lysis wasaccomplished by resuspending the cell pellets in 300-700 μl Tris-HClbuffer (100 mM, pH 8.2) containing lysozyme (0.5-1 mg/ml) anddeoxyribonuclease I (1.5-4 Units/ml). The pellets were resuspended andlysed by mixing using a Beckman Multimek 96-channel pipetting robot forapproximately 15 minutes before centrifugation. An appropriate volume(10-50 μl) of the resulting cell lysates containing soluble P450 hemedomain mutants were used in the activity assay.

All enzyme activity measurements using p-nitrophenoxy-derivativesubstrates were performed by monitoring the formation ofp-nitrophenolate (pNP) (398 nm) at room temperature using a 96-wellplate spectrophotometer (SPECTRAmax, Molecular Devices). A typicalreaction in a well contained 130 μl 100 mM Tris-HCl buffer pH 8.2, 10 μlstock solution of substrate in DMSO, and 10 μl enzyme solution (purifiedor as lysate). Reactions were initiated by the addition of 10 μl H₂O₂stock solution. Typical final concentrations were 250 μM substrate(12-pNCA), 1-50 mM H₂O₂, and 0.1-1.0 μM P450.

The 398 nm absorbance reading for each well was blanked before additionof H₂O₂ so that end point turnovers could be calculated. Rates ofperoxide shunt pathway activity for the mutants were calculated as therate of pNP formation over time (or the increase in absorbance at 398 nmover time). The value for (extinction coefficient)*(path length) for pNPunder the exact conditions used in the spectrophotometer assay wascalculated from a standard curve generated with known concentrations ofpNP. This factor was used to quantify turnover of substrate. The DMSOconcentrations used were shown to have no significant effect on theextinction coefficient of pNP.

The most active mutants in a generation were streaked out on agar platesto obtain single colonies. Single colonies were then picked forrescreening. Rescreening was performed as described above, except 10 mlTB cultures were grown instead of deep-well plate cultures. Cell pelletsfrom the centrifuged 10 ml TB cultures were resuspended in 1 ml Tris-HCl(100 mM, pH 8.2) and lysed by sonication. Cell lysates were centrifugedand P450 concentrations in the lysates were then quantified byCO-binding. Specific activities and total enzyme turnover values werethen determined to verify that the selected mutants indeed showedimproved activity over the parent enzyme. Specific activity is definedas moles of product formed/mole of P450/minute, where product is pNP,quantified by the absorbance at 398 nm. Total turnover is defined as thetotal number of moles of product produced per mole of enzyme.

Screening for Thermostability

Screening for thermostability was accomplished in the same manner asscreening for activity, with the addition of a heat inactivation step.After the activities of the lysates from a deep-well plate have beenscreened as described above, 50 μl aliquots of each lysate were pipettedfrom the plate and into a 96-well PCR plate (GeneMate). These aliquotswere heated to an appropriate temperature (48° C.-56° C.) in a PTC200thermocycler (MJ Research) for 10-15 minutes, rapidly cooled to 4° C.,and then brought to room temperature. The residual activities of theseheat-inactivated lysates were then measured in the same manner that theinitial activities were measured. Thermostability was defined as thefraction of initial activity remaining after the heat inactivation.Incubation temperatures were chosen so that the parent of a generationof mutants retained 20%-30% of its residual activity. As examples, themutant library that was generated with mutant 21B3 as the parent genewas screened by heating to 48.5° C. for 10 minutes. The mutant librarythat resulted in thermostable mutant TH4 was screened by heating to 56°C. for 15 minutes. Criteria for selection of mutants was that they beboth more thermostable than their parent, and able to maintain the same(or nearly the same) peroxide shunt pathway activity as the parent.

General Assay for Measuring P450 Activity

In general, and unless otherwise stated, enzyme activities were measuredusing p-nitrophenoxy-derivative substrates (e.g. 12-pNCA) by monitoringthe formation of p-nitrophenolate (pNP) (398 nm) at room temperatureusing a 96-well plate spectrophotometer (SPECTRAmax, Molecular Devices),as described above. Typical reactions in a well contained 130 μl 100 mMTris-HCl buffer pH 8.2, 10 μl stock solution of substrate (e.g. 4 mM12-pNCA) in DMSO, and 10 μl enzyme solution (purified or as lysate).Peroxide shunt pathway activities were measured by the addition of H₂O₂(1-50 mM), while NADPH-driven hydroxylation by full length P450 enzymeswas measured by addition of NADPH (0.2-1 mM).

Quantification of enzyme rates and total turnover numbers were performedas described above. Briefly, P450 enzyme concentrations were determinedby CO-binding. Product concentrations were determined as theconcentration of para-nitrophenolate (pNP) produced in a well, which wasdetermined from standard curves prepared by varying concentrations ofpNP and recording the absorbance at 398 nm. Initial rates weredetermined as the rate of pNP formation in the first few seconds of thereaction, before there was any noticeable change in reaction rate.

Purification of P450 BM-3 Variants

Purification of full-length wild-type P450 BM-3 and full length P450BM-3 F87A was performed essentially as described (Schwaneberg et al.,1999(b)) using an Äkta explorer system (Pharmacia Biotech) andSuperQ-650M column packing (Toyopearl).

Purification of the heme domain enzymes took advantage of the 6-Hissequence cloned into the C-terminus of each enzyme by using theQIAexpressionist kit (Qiagen) for purification under native conditions.Briefly, cultures were grown for protein expression, as described above.Cells were centrifuged, resuspended in lysis buffer (10 mM imidazole, 50mM NaH2PO4, pH 8.0, 300 mM NaCl), and lysed by sonication. Cell lysateswere centrifuged, filtered, and loaded onto Qiagen Ni-NTA column. Thecolumn was washed with wash buffer (20 mM imidazole, 50 mM NaH2PO4, pH8.0, 300 mM NaCl), and the bound P450 was then eluted with elutionbuffer (200 mM imidazole, 50 mM NaH2PO4, pH 8.0,300 mM NaCl).

Aliquots of the purified protein were placed into liquid nitrogen andstored at −80° C. When used, the frozen aliquots were rapidly thawed andbuffer-exchanged with 100 mM Tris-HCl, pH 8.2 using a PD-10 Desaltingcolumn (Amersham Pharmacia Biotech). P450 concentrations were thendetermined by the CO-binding difference spectrum.

Determination of shunt pathway activity and product distributions withmyristic acid, lauric acid, decanoic acid, and styrene.

A typical reaction contained 1-4 μM purified P450 heme domain enzyme and1-2 mM substrate in 500 μl 100 mM Tris-HCl, pH 8.2 (for reactions withstyrene the solution also contained 1% DMSO). Reactions were initiatedby the addition of 1-10 mM H₂O₂. For determining rates, the reactionswere stopped at specific time points (e.g., 1, 2, and 4 minutes) by theaddition of 7.5 μl 6 M HCl for the reactions on fatty acids. Reactionsusing styrene as substrate were stopped by the addition of 1 ml pentanefollowed by vigorous shaking. For determining total turnover values, thereactions were allowed to continue until the enzyme was completelyinactivated by the peroxide. At the end of each reaction an internalstandard was added prior to extraction. For reactions with myristic andlauric acid, 30 nmoles of 10-hydroxydecanoic acid was used as theinternal standard. For reactions with dodecanoic acid, 30 nmoles of12-hydroxylauric acid was added the internal standard. Finally, 200nmoles of 3-chlorostyrene oxide was added as the internal standard forstyrene reactions.

Reactions with styrene were extracted twice with 1 ml pentane. Thepentane layer was evaporated down to ˜200 μl to concentrate theproducts. Fatty acid reactions were extracted twice with 1 ml ethylacetate. The ethyl acetate layer was dried with sodium sulfate and thenevaporated to dryness in a vacuum centrifuge. The resulting productresidue was dissolved in 100 μl of a 1:1 pyridine:BSTFA(bis-(trimethylsilyl-trifluoroacetamide) mixture containing 1%trimethylchlorosilane (TMCS). This mixture was heated at 80° C. for 30minutes to allow for complete derivitization of the acid and alcoholgroups to their respective trimethylsilyl esters and ethers.

Reaction products were identified by GC/MS using a Hewlett Packard 5890Series II gas chromatograph coupled with a Hewlett Packard 5989A massspectrometer. Quantification of lauric acid, decanoic acid, and styrenereaction products was accomplished using a Hewlett Packard 5890 SeriesII Plus gas chromatograph equipped with a flame ionization detector(FID). The GCs were fitted with an HP-5 column. Authentic standards foreach hydroxylated isomer of the fatty acids were not available, sostandard curves were generated using the available ω-hydroxylatedstandards (12-hydroxylauric acid and 10-hydrodecanoic acid). Authenticstandard samples were prepared in the same fashion as the reactionsamples, except the enzyme was inactivated by the addition of HCl beforethe addition of peroxide. All peak areas were normalized by dividing bythe peak area of the internal standard added to each sample. It wasassumed that the FID response is the same for all regioisomers of agiven hydroxylated fatty acid. For styrene, the only product detectedwas styrene oxide, for which the authentic standard was available.

Reactions that were stopped one minute after the addition of peroxidewere used to estimate the initial rates of peroxide shunt pathwayactivity on each substrate. The quantity of product in the reactionmixture was determined from the standard curve and divided by thequantity of P450 present in the reaction, giving an estimate of theinitial rate (nmol product/nmol P450/min).

Results

Both wild-type BM-3 and the F87A mutant were tested for shunt pathwayactivity using 12-pNCA as substrate. Whereas H₂O₂-driven activity couldnot be detected with the wild-type BM-3, the F87A mutant was able to useH₂O₂ for 12-pNCA hydroxylation at detectable levels (˜50 nmolproduct/mol P450/min when using 10 mM H₂O₂ and ˜90 nmol product/nmolP450/min using 50 mM H₂O₂). The Km,app of BM-3 F87A for H₂O₂ wasestimated to be ˜15 mM using enzyme from lysates. The enzyme is veryshort-lived in the presence of peroxide: in 50 mM H₂O₂ most activity islost after ˜2 minutes.

A comparison of NADPH-driven versus H₂O₂-driven activity in cell lysatescontaining BM-3 F87A showed that shunt pathway activity was retained forlonger periods than NADPH activity. Whereas less than 10% of thelysate's NADPH activity remained after sitting one day at roomtemperature, the same lysate retained more than 63% of the shunt pathwayactivity. This is likely to be due to the labile link between the hemedomain and the reductase domain. This may also be in part due to agreater instability of the reductase domain compared to the heme domain,or a greater instability of one or more protein components involved inthe electron transfer process used by the NADPH pathway compared to theheme domain. Regardless, this is strong evidence that it is easier toengineer stability in the heme domain alone than in the full length BM-3enzyme.

When using hydrogen peroxide instead of NADPH, the reductase domain ofP450 BM-3 is not necessary and only places an added burden on the E.coli host during protein expression. Therefore a nucleotide sequenceencoding the heme domain alone was cloned into the pCWori+ vector, whichwas found to result in approximately four-fold higher molar expression.

The P450 BM-3 heme domain was considered to be composed of the first 463amino acids of the full-length BM-3 protein (not including the startmethionine, which is considered to be amino acid numbered zero). Thesequence coding for six histidines was cloned onto the end of the BM-3heme domain gene, resulting in a 469 amino acid protein. P450 hemedomain mutant F87A containing a 6-His tag was chosen as the startingpoint for directed evolution experiments. That is, the gene coding forthis variant served as parent template used for generating the firstmutant library to be screened for improvements in shunt pathwayactivity. The addition of the 6-His tag had a negligible effect on shuntpathway activity for the F87A mutant.

E. coli naturally produces catalase and the presence of catalase in thelysate was problematic in the development of a screening assay for shuntpathway activity. Bubbles were formed from the catalase reaction, andH₂O₂ concentrations were rapidly reduced. Therefore a catalase-free E.coli strain was used, in which the genes that code for catalase wereknocked out of the host genome (Nakagawa et al., 1996). This strainprevented bubble formation, and allowed maintaining steadyconcentrations of H₂O₂, resulting in a sensitive screening system.

As described above, P450 BM-3 heme domain mutant F87A (F87A mutation inSEQ ID NO:3) was used as the starting point for directed evolution ofH₂O₂-driven hydroxylation of the surrogate substrate 12p-nitrophenoxy-carboxylic acid (12-pNCA). Mutant libraries were screenedfor activity in both 1 mM H₂O₂ and 50 mM H₂O₂ in efforts to improveactivity and stability in H₂O₂. Mutagenesis by error-prone PCR andscreening generated F87A heme domain variants with up to five-foldimproved total-shunt pathway activity. Generating heme domains or thefull length enzyme makes no difference since the shunt pathway activityis the same, and the reductase portion has no influence.

The first generation resulted in mutants “2H1”, “1F8” and “2E10”. Twoseparate second generation libraries were then created and screened,resulting in mutants “1F8-1” and “1F8-2” (where “1F8” was the parentgene), and “2E10-1”, “2E10-2”, “2E10-3”, and “2E10-4” (where “2E10” wasthe parent gene).

Mutant 2E10-1 had an initial rate of ˜50 nmol/nmol P450/min in 1 mMH₂O₂, while the rate with F87A is ˜10 nmol/nmol P450/min. Sequencing ofseveral improved variants revealed a number of mutations that conferthese improvements. The mutants and known mutations are listed in Table4. TABLE 4 Mutations from error-prone PCR resulting in BM-3 heme domainvariants showing improved H₂O₂-driven hydroxylation. All mutantsadditionally comprise the F87A substitution. Variant where Mutation BaseChange Amino Acid Change First Appears A26T K9I 1F8 A213G (SILENT) 2H1A278G E93G 2E10-3* A299G H100R 1F8 A337G K113E 2E10 A650T N186S 2E10-3*A650T D217V 2E10-1 A709T M237L 2E10-4* A731G E244G 1F8 G735A (SILENT)1F8 A885G (SILENT) 2E10-3* T1188A (SILENT) 2E10 A1300G K434E 2E10 and2H1*Parent is 2E10

Mutation K434E was noted to have appeared in two separately evolvedmutants (“2H1” and “2E10”), indicating that this mutation is especiallyeffective in improving peroxide shunt activity. Additional improvedmutants include 1F8-1 and 1F8-2 (whose parent is 1F8) and 2E10-2 (whoseparent is 2E10).

Example 2 Improved Hydrogen Peroxide-Driven Hydroxylation by EvolvedCytochrome P450 BM-3 Heme Domain

This Example describes the discovery of novel cytochrome P450 BM-3variants that use hydrogen peroxide (H₂O₂) for substrate hydroxylationmore efficiently than the wild-type enzyme.

Materials and Methods

The same materials and methods were used in this Example as thosedescribed in Example 1. However, in Example 2, StEP recombination wascarried out with error-prone mutants. A 50 μl PCR reaction contained˜160 ng total template DNA (comprised of approximately equalconcentrations of the seven mutant genes), 0.2 mM dNTPs, 5 pmole outsideprimers, 5 μl Qiagen Hotstar buffer (containing 15 mM Mg²⁺), and 2.5 UHotstarTaq polymerase. PCR was performed in a PTC200 thermocycler (MJResearch). The temperature protocol was as follows: (hot start) 95° C.for 3 min, followed by 100 cycles of 94° C. for 30 sec and 58° C. for 8sec. Genes from seven mutants were used and resulted in someimprovements.

Results

One round of StEP recombination (Zhao et al., 1999) was performed, whichresulted in mutants “stepB6” and “stepB3”. StEP recombination wasperformed essentially as described (Zhao et al., 1999) using HotStarTaqDNA Polymerase (Qiagen). The parent genes used for the recombinationincluded variants “2H1”, “1F8-1”, “1F8-2”, “2E10-1”, “2E10-2”, “2E10-3”,AND “2E10-4”.

Mutant libraries were screened for activity on the surrogate substrate12-p-nitrophenoxy-carboxylic acid (12-pNCA) in both 1 mM H₂O₂ and 50 mMH₂O₂. A combination of error-prone PCR and recombination of improvedmutants by staggered extension process (StEP) resulted in variants withimproved shunt pathway activity. Mutant “stepB3” had a total activitythat was seven-fold higher than the BM-3 F87A mutant in 50 mM H₂O₂ and atotal turnover in 1 mM H₂O₂ that was four times higher than F87A.Sequencing of this mutant revealed five mutations in the DNA sequence,corresponding to four amino acid changes (see Table 5).

Another variant found in the StEP library, “stepB6”, showed similaractivity to “stepB3”, but has a lower apparent Km for H₂O₂ (about 8 mM)and has CO-binding difference spectrum peaks at both 450 nm and 420 nm.This spectral property is typically indicative of a misfolded andinactive P450, and indicates a change in the electron character of theproximal ligand. The 420 nm CO-binding peak has been observed with otherheme enzymes that more readily bind H₂O₂ (e.g., peroxidases). Thesequence of “step B6” was only one amino acid change different from“stepB3”. The mutations are listed in Table 5.

One goal of this experiment was to combine the properties of a mutantactive at high peroxide concentrations with the properties of anothermutant active at low peroxide levels. This indeed worked. Mutant“stepB6” showed improved activity under both conditions: more thansix-times faster than the F87A mutant in 1 mM H₂O₂ and more thanfive-fold higher total turnover than F87A in 50 mM H₂O₂. TABLE 5Mutations in “stepB3” and “stepB6” P450 BM-3 variants (in addition toF87A) Amino Acid Base Substitution Substitution Step B3 Step B6 A299GH100R X X A433G M145V X X A709T M237L — X T820A S274T X — T1188A(SILENT) X X A1300G K434E X X

The mutations in the step B3 and B6 variants were recognized asparticularly important for improved peroxide-utilization, since thesemutations were present in products of recombination, whereby the pointmutations of seven different mutants (each with different pointmutations accumulated from previous rounds of error-prone PCR) wereallowed to assemble in all possible combinations. In this manner it iseasy to screen for and isolate improved recombinant products with onlybeneficial or neutral mutations, and all deleterious mutations removed.

Example 3 Improved Peroxide-Driven Hydroxylation by Evolved CytochromeP450 BM-3 Heme Domain

This Example describes a novel cytochrome P450 BM-3 variant that usehydrogen peroxide (H₂O₂) for substrate hydroxylation more efficientlythan the wild-type enzyme.

Methods and Results

Further rounds of directed evolution to improve peroxide shunt pathwayactivity were carried out starting with mutant “stepB3”. Error-prone PCRwas used to generate mutant libraries, and screening was performed asdescribed above using 1 mM H₂O₂. After two rounds of evolution mutant“21B3” was isolated.

After reacting wild-type, F87A and 21B3 with laurate, the reactionproducts were extracted, dried, and derivatized to the trimethylsilylesters and ethers. The regiospecificity was quite different for thewild-type compared to F87A and 21B3. The F87A mutation appears tobroaden regiospecificity and shift hydroxylation away from the terminalpositions. Whereas the wild-type BM-3 typically oxidizes fatty acidsexclusively at positions ω-1, ω-2, and ω-3 under the NADPH pathway (aswell as under the peroxide shunt pathway, although at much lowerlevels), mutant F87A hydroxylates fatty acids at positions ω-1, ω-2,ω-3, ω-4, and ω-5 under the NADPH and peroxide shunt pathways. GCanalysis of the reaction mixture showed that the total product arearelative to the internal standard (IS) area for 21B3, heme domain mutantF87A, and wild-type was 8.9, 1.1, and 0.11, respectively. The relativeratios of the hydroxylated positions varies with the substrate andappears to be the same in evolved mutants “21B3” and “TH4”, whichcontain the F87A mutation. Sequencing of mutant 21B3 revealed 13mutations in the DNA sequence, corresponding to 9 amino acid changes (inaddition to F87A). The mutations are listed in Table 6. TABLE 6Mutations in peroxide-dependent mutant “21B3” (in addition to F87A).Base Change Amino Acid Change A172G I58V A195T (SILENT) A299G H100RC321A F107L G403T A135S A433G M145V A684G (SILENT) A715C N239H T810C(SILENT) T820A S274T T1188A (SILENT) A130OG K434E G1336A V446I

For characterization, enzymes were purified by binding the 6-His tag toa Ni-NTA agarose column (Qiagen), washing, and eluting with imidazole(as described above). The imidazole was then removed in a bufferexchange column. Mutant “21B3” was found to be more than fifteen timesmore active than mutant F87A on 12-pNCA using 5 mM H₂O₂ (490 nmol/nmolP450/min versus 30 nmol/nmol P450/min). The total turnover of 12-pNCAachieved by mutant “21B3” was approximately twelve times higher thanmutant F87A (˜1000 versus ˜80 in 5 mM H₂O₂).

Similar improvements in activity were seen with real fatty acidsubstrates by GC analysis. Using laurate (dodecanoic acid) and 5 mMH₂O₂, mutant 21B3 was approximately eight times more active than F87A(˜28 nmol/nmol P450/min vs. ˜3 nmol/nmol P450/min using 10 mM H₂O₂). TheGC data indicated that wild-type BM-3 is capable of only single toperhaps triple total turnovers under the shunt pathway.

Similar activity results were also found with myristic acid, decanoicacid, and styrene. Decanoic acid was oxidized by “21B3” at an initialrate of ˜82 nmol/nmol P450/min, whereas the initial rate with F87A was˜10 nmol/nmol P450/min using 10 mM H₂O₂. Finally, the peroxide-drivenoxidation of styrene to styrene oxide by “21B3” had an initial rate of˜50 nmol/nmol P450/min using 10 mM H₂O₂, while the rate with F87A wasnot detectable. It should be noted that the shunt pathway activity ofmutant “21B3” on styrene is higher than the normal NADPH-driven activityof wild-type BM-3 on this same substrate (˜30 nmol/nmol P450/min using0.2 mM NADPH).

The initial 12-pNCA hydroxylation rate for P450 BM-3 variant 21B3 atvarious peroxide concentrations was compared to that of the F87A variantand wild-type enzyme heme domains. The same results have been verifiedwith the full protein, as described in the Materials and Methodssection. The 21B3 heme domain variant was found to yield a peak initial12-pNCA conversion rate of 780 mole product per mole enzyme per minuteat 25 mM H₂O₂, whereas the initial rates for the F87A heme domain atthis peroxide concentration was only 76 mole product per mole enzyme perminute. The rates for wild-type BM-3 were not detectable.

In addition, the total turnover of 12-pNCA of 21B3 in the peroxide shuntpathway was compared to the corresponding F87A and wild-type enzymes atvarious concentrations of H₂O₂. This assay was carried out as describedabove (see Materials and Methods). At concentrations of 1, 5, and 10 mMH₂O₂, the total substrate turnover of 21B3 was about 17, 12, and 10times higher than the F87A variant, whereas the total turnover of thewild-type enzyme was barely distinguishable. The turnover units aretotal moles of product made per mole of P450 up to the point that it haslost all activity.

EXAMPLE 4 Peroxide-Dependent, Thermostable Cytochrome P450 BM-3 Variants

It was noticed that the stability of the evolved peroxide-driven mutantswas lower than that of the original F87A parent. Stability of thesemutants is an important factor when considering possible applications.Mutants with greater thermostability could be used at elevatedtemperatures and would potentially have even greater activity atelevated temperatures. Therefore this example sought to improve thethermostability of the peroxide-dependent mutants without sacrificingactivity.

Starting with mutant “21B3”, directed evolution to improvethermostability while retaining maximum peroxide shunt pathway activitywas performed using error-prone PCR to generate mutant libraries.Libraries were screened using 1-5 mM H₂O₂. After screening threegenerations of libraries created with error-prone PCR (as describedabove), thermostable mutant “TH3” was isolated. An additional librarywas generated with “TH3” as the parent using the GeneMorph PCRMutagenesis Kit (Stratagene), resulting in thermostable mutant “TH4”.TABLE 7 Mutations in peroxide-dependent, thermostable P450 BM-3 variant“TH4”, in addition to F87A. (Percentage values represent the changes incodon usage by E. Coli) Base Change(s) Amino Acid Change A172G I58VA195T SILENT (S); 14% to 15% A299G H100R C321A F107L G403T A135S A433G +T434C M145A A684G SILENT (E); 67% to 33% A715C N239H T810C SILENT (S);16% to 26% T820A S274T T970A L324I A1096G I366V T1188A SILENT (G); 33%to 13% A1300G K434E T1309C SILENT (L); 14% to 4% G1324A E442K G1336AV446I

The only difference between the mutations in TH4 and the mutations inthe mutant from the previous generation (mutant “TH3”, which was theparent used to generate the library that resulted in TH4) is thatpreviously occurring mutation M145V was changed to M145A. Thus,throughout the course of evolving shunt pathway activity and stability,a single codon was mutated on two separate occasions, resulting in anamino acid (Ala) that could not be reached by a single base mutation.

The thermostability of the TH4 variant was compared to the 21B3 and F87AP450 BM-3 variants by comparing the ratios of residual activity toinitial activity of each enzyme after incubation at various temperaturesin the range of 35-65° C. for 10 minutes. Activities before and afterheat inactivation were measured using H₂O₂ and 12-pNCA as described inthe Methods. This test was conducted in the absence of cofactor. Theresults showed that TH4 retained activity to a higher degree than F87Avariant, which, in turn, was more stable than 21B3. Additionally, TH4had essentially the same initial activity as “21B3”. Thus, of theseenzyme variants, TH4 was most thermostable (at least as stable as theoriginal parent (F87A)), and retained peroxide activity essentiallyequal to that of 21B3. Because of its stability, TH4 has a greaterapplicability for higher temperature environments, where its activitywill also be higher. The mutations that appear to play a particular rolein thermostability are therefore M145A, L324I, I366V, and E442K (thosewhich have been accumulated throughout the thermostability directedevolution process).

Different peroxides were also tested, including cumene hydroperoxide,t-butyl hydroperoxide, and peracetic acid, for their utilization by theP450 BM-3 variants. Of the different peroxides, H₂O₂ was found to bemost effective in the 12-pNCA assay, where 12-pCNA is hydroxylated atC-12, followed by peracetic acid, for both the BM-3 F87A mutant and theevolved variants.

EXAMPLE 5 Thermostable P450 BM-3 Peroxygenase Variants

The laboratory-evolved P450 BM-3 heme domain variant TH4, which hassignificantly improved peroxygenase activity (H₂O₂-driven hydroxylation)compared to the wild-type enzyme, and improved peroxygenase activity aswell as thermostability as compared to the heme domain of the F87Amutant (HF87A), is described above. This Example describes furtherimproving thermostability to a level better than the wild-type enzymeheme region without sacrificing the improved peroxygenase activity overthe wild-type enzyme.

Methods

General Remarks. All chemical reagents were procured from Aldrich,Sigma, or Fluka. Restriction enzymes were purchased from New EnglandBiolabs and Roche. Deep-well plates (96 wells, 1 ml volume per well) forgrowing mutant libraries were purchased from Becton Dickinson.Flat-bottom 96-well microplates (300 μl per well) for screening mutantlibrary activities were purchased from Rainin.

Enzyme Expression and Purification. P450 BM-3 enzymes were expressed incatalase-deficient E. coli (Nakagawa et al., 1996) using theα-D-thiogalactopyranoside (IPTG)-inducible pCWori+ vector (Barnes etal., 1991). The heme domain consisted of the first 463 amino acids ofP450 BM-3 followed by a 6-His sequence at the C-terminus, which had nosignificant influence on activity. Cultures for protein production weregrown and proteins were purified as described (Cirino and Arnold,2002(a)). Purified enzyme samples were stored at −80° C. until use, atwhich time they were thawed at room temperature and then kept on ice.Concentrations of properly-folded P450 enzyme were determining from the450 nm CO-binding difference spectra of the reduced heme, as described(Omura and Sato, 1964).

Preparation of Mutant Libraries. Error-prone PCR libraries were preparedusing standard protocols (Cirino et al., 2003). Starting with 21B3 asthe parent, three rounds of error-prone PCR (using Taq DNA polymerase(Roche)) followed by screening were performed, and the most thermostablemutant which did not lose peroxygenase activity was chosen as the parentfor the next generation. Two additional generations were prepared withthe GeneMorph™ PCR Mutagenesis Kit (Stratagene). In the final generationleading to mutant 5H6, a recombinant library was prepared by DNAshuffling (Stemmer, 1994) using Pfu Ultra DNA Polymerase™ (Stratagene).Parents for the recombinant library included HF87A, mutants from theprevious generation which were more stable but less active, and mutantswith increased activity.

Mutant Library Screening. Screening was performed as described below,subjecting cell lysates to a heat inactivation step and screening forresidual activity (see also (Cirino and Georgescu, 2003)). Briefly,cultures expressing mutants were grown in 96-well deep-well plates.After cell growth, the plates were centrifuged, cell pellets were frozenat −20° C., and the cells were lysed in Tris-HCl buffer (100 mM, pH 8.2)containing lysozyme (0.5-1 mg/ml) and deoxyribonuclease 1 (1.5-4Units/ml). Clarified cell lysates were transferred to 96-wellmicroplates for activity measurements at room temperature (describedbelow). Lysates were also transferred to 96-well PCR plates (GeneMate)and heated to an appropriate temperature (48° C.-57.5° C.) in a PTC200thermocycler (MJ Research) for 10-15 minutes, rapidly cooled to 4° C.,and then brought to room temperature. The residual activities of theseheat-treated lysates were then measured in the same manner as theinitial activities. Clones showing a higher fraction of activityremaining after heat treatment and high initial activity werecharacterized further.

Activity Assay. Activity on 12-pNCA (Schwaneberg et al., 1999) wasdetermined by monitoring the formation of p-nitrophenolate (pNP) (398nm) at room temperature using a 96-well plate spectrophotometer(SPECTRAmax Plus, Molecular Devices), as described. Reaction wellscontained Tris-HCl buffer (140 μl of 100 mM, pH 8.2), a stock solutionof substrate (10 μl of 4 mM 12-pNCA) in DMSO, and purified enzyme orclarified lysate. Reactions were initiated by the addition of an H₂O₂stock solution (10 μl). Data for accurate determination of 12-pNCAturnover rates with purified enzyme were collected using a BioSpec1601spectrophotometer (Shimadzu), where absorbance changes could beregistered every 0.1 seconds. Typical final concentrations were 250 μM12-pNCA, 6% DMSO, 1-10 mM H₂O₂, and 0.1-1.0 μM P450. The extinctioncoefficient for pNP was determined from standard pNP solutions preparedunder identical reaction conditions. NADPH-driven activity of BWT wasdetermined spectrophotometrically from the initial rate of NADPHconsumption (measured as the decrease in 340 nm absorbance) in thepresence of myristic acid, as described (Yeom and Sligar, 1997).

Data for T₅₀ Determination. Purified enzyme samples (˜20 μM) in Tris-HClbuffer (100 mM, pH 8.2) were incubated for 10 minutes at differenttemperatures. Samples were then cooled on ice, and the concentration ofproperly-folded heme domain (diluted 8×) was estimated from the 450 nmCO-binding difference spectra and compared to the CO-binding peak priorto heat treatment. Residual NADPH-consumption activity was measured forBWT. Data in FIG. 8 represent average values from at least twoexperiments.

Data for t½ Determination. Concentrated purified enzyme (70 μM) wasadded to pre-heated (57.5° C.) Tris-HCl buffer (100 mM, pH 8.2) andincubated at 57.5° C. Samples were removed at time intervals, quenchedby dilution into cold buffer, brought to room temperature, and assayedfor residual activity. Data in FIG. 9 represent average values from atleast two experiments.

Results

TH4 was used as the parent of a random mutagenesis library. Since novariants which were both more stable and more active than TH4 wereidentified in this first library, the genes of mutants which were eithermore active or more thermostable were recombined using DNA shuffling toproduce a recombinant library. Screening the recombinant libraryresulted in thermostable variant 5H6. TABLE 8 Thermostability andactivity parameters for evolved and parental P450s. BWT = full-length,wildtype P450 BM-3; HWT = wildtype P450 BM-3 heme domain; HF87A = P450BM-3 heme domain containing mutation F87A; 21B3 & 5H6 = evolved hemedomain peroxygenase variants. T₅₀ for 10-minute Peroxygenaseincubations^([a]) t½ at 57.5° C.^([b]) Activity^([c]) Mutant (° C.)(minutes) (minute⁻¹) BWT 43 0.46 <5 HWT 57 n.d. <5 HF87A 54 2.3 23 21B346 n.d. 430 5H6 61 115 220^([a])Calculated from the data in FIG. 8, fit to two-state denaturationequation.^([b])Calculated from the data in FIG. 9, fit to a first-orderexponential decay equation.^([c])Reported as initial rates at room temperature on 12-pNCA in 10 mMH₂O₂ and 6% DMSO.n.d.: not determined.

According to this measure of stability, variant 5H6 (T₅₀=61° C.) is morethermostable than the natural catalytic system, BWT (T₅₀=43° C.) and thewild-type heme domain (T₅₀=57° C.). It is also significantly morethermostable than HF87A and 21B3. The substitutions found in 5H6 arelisted in Table 9, and are depicted in FIG. 6C. TABLE 9 Mutations inthermostable peroxygenase variant 5H6, in addition to F87A. (Percentagevalues represent the changes in codon usage by E. coli) Base Change(s)Amino Acid Change T154A L52I A172G I58V A195T SILENT (S); 14% to 15%A299G H100R C318G S106R C321A F107L G403T A135S C489T SILENT (N); 52% to48% C551T A184V A684G SILENT (E); 67% to 33% A715C N239H T810C SILENT(S); 16% to 26% T820A S274T T970A L324I G1018A V340M A1096G I366V T1188ASILENT (G); 33% to 13% A1300G K434E T1309C SILENT (L); 14% to 4% G1324AE442K G1336A V446I CAT (1405, 1406, 1407) DELETED H469 DELETED

Throughout the course of evolving shunt pathway activity and stability,the codon for residue position 145 was changed on two separateoccasions: from ATG to GTG in 21B3 (mutation M145V) and then to GCG(mutation M145A, which could not be reached by a single base mutation).This mutation was removed during DNA shuffling, resulting in mutant 5H6.

Thermostable peroxygenase 5H6 contains five new amino acid substitutionscompared to TH4, which includes the reversion of M145A back to M145:L521, S106R, M145, A184V, and V340M. 5H6 also contains a deletionresulting in the removal of one His residue from the 6-His sequenceincluded at the C-terminus. Substitutions L521, A184V, and V340M areconservative with regard to hydrophobicity and size. The serine residueat position 106 was converted to a positively charged Arg residue(S106R). These mutations increased the enzyme's stability. According tothe P450 BM-3 heme domain crystal structure, substitutions S106R andV340M are located on the protein surface; the others are buried.

Altogether, four thermostabilizing mutations are close to positionswhere mutations that improved peroxygenase activity accumulated inearlier experiments: L521 (in β-sheet 1-2) is adjacent to I58V (helix B)from 21B3, S106R (in a loop connecting helices C and D) lies next tomutation F107L from 21B3, E442K (in β-sheet 4-2) lies adjacent to K434E(in β-sheet 4-1) from 21B3, and the reversion to M145 (helix E) isadjacent to S274T (helix I) from 21B3. See FIG. 7. Without being boundto any specific theory, the new stabilizing mutations may thereforealleviate structural perturbations introduced by the original mutationswhich improved peroxygenase activity.

Enzyme thermostabilization can lead to a shift in theactivity-temperature profile to higher temperatures, reflecting thehigher stability of the folded protein (Daniel et al., 2001).Measurements of peroxygenase activity at different temperatures,however, showed no significant increase in the optimum temperature foractivity for 5H6 compared to HF87A (both were in the range 25-30° C.).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application and in the appendedbibliography, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

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1. An isolated variant of a cytochrome P450 BM-3 comprising the aminoacid sequence of SEQ ID NO:3, the variant comprising at least a firstmutation in an amino acid residue selected from K9, I58, F87, E93,H1100, F107, K113, A135, M145, 145A, A184, N186, D217, M237, E244, S274,L324, 1366, K434, E442, and V446 of SEQ ID NO:3, and at least a secondmutation in an amino acid residue selected from L52, S106, N239, andV340.
 2. The isolated variant of claim 1, comprising mutations in atleast three amino acid residue selected from K9, I58, F87, E93, H100,F107, K113, A135, M145, 145A, A184, N186, D217, M237, E244, S274, L324,1366, K434, E442, and V446 of SEQ ID NO:3, and mutations in at leastthree amino acid residues selected from L52, S106, N239, and V340. 3.The isolated variant of claim 1, comprising a mutation in L52, I58, F87,H100, S106, F107, A135, A184, N239, S274, L324, V340, 1366, K434, E442,and V446.
 4. The isolated variant of claim 1, wherein the cytochromeP450 BM-3 does not include a reductase domain.
 5. The isolated variantof claim 1, wherein the variant has a higher thermostability than P450BM-3.
 6. The isolated variant of claim 1, wherein the variant has ahigher thermostability than the cythchrome P450 BM-3 heme domain.
 7. Theisolated variant of claim 1, wherein the first mutation is selected fromK91, 158V, F87A, F87S, E93G, H100R, F107L, K113E, A135S, M145A, M145V,A184V, N186S, D217V, M237L, E244G, S274T, L3241, 1366V, K434E, E442K,and V446I.
 8. The isolated variant of claim 1, wherein the secondmutation is selected from L52I, S106R, N239H, and V340M.
 9. The isolatedvariant of claim 1, wherein the variant comprises at least 6 mutationsselected from K9I, L52I, I58V, F87A, F87S, E93G, H100R, S106R, F107L,K113E, A135S, M145A, M145V, A184V, N186S, D217V, M237L, N239H, E244G,S274T, L3241, V340M, 1366V, K434E, E442K, and V4461.
 10. The isolatedvariant of claim 1, wherein the variant comprises the mutations L521,158V, F87A, H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I,V340M, I366V, K434E, E442K, and V446I.
 11. A method of thermostabilizinga parent cytochrome P450 oxygenase heme domain having at least a firstmutation in a wild-type cytochrome P450 oxygenase heme domain, themethod comprising: (a) preparing a protein library of variants of theparent having at least a second mutation, which second mutation islocated no more than 10 Ångströms from the first mutation; and (b)selecting any variant having a higher thermostability than the parent.12. The method of claim 11, wherein selecting any variant comprisesselecting any variants having a T50 higher than that of the parentcytochrome P450 oxygenase heme domain.
 13. The method of claim 11,wherein selecting any variant comprises selecting any variants having aT50 higher than that of the wild-type cytochrome P450 oxygenase hemedomain.
 14. The method of claim 11, wherein the parent cytochrome P450oxygenase heme domain comprises an amino acid sequence at least 95%identical to SEQ ID NO:3.
 15. The method of claim 11, wherein the firstmutation is in at least one amino acid selected of the group consistingof I58, H100, F107, A135, M145, N239, S274, K434, and V446.
 16. Themethod of claim 15, wherein the first mutation is at least one aminoacid substitution selected from the group consisting of I58V, H100R,F107L, A135S, M145V, M145A, N239H, S274T, K434E, and V446I.
 17. Themethod of claim 11, wherein the second mutation is in at least one aminoacid selected from the group consisting of L52, S106, M145, L324, I366,and E442.
 18. The method of claim 17, wherein the second mutation is atleast one amino acid substitution selected from the group consisting ofL521, S106R, M145A, L3241, 1366V, E442K, and a reversal of a mutation inM145.
 19. The method of claim 18, wherein the second mutation is atleast one amino acid substitution selected from L52I, S106R, and E442K.20. An isolated variant of a parent cytochrome P450 oxygenase hemedomain, the parent comprising at least a first mutation in a wild-typecytochrome P450 oxygenase heme domain and having at least 90% sequenceidentity to SEQ ID NO:3; and the variant comprising at least a secondmutation no more than 10 Angstroms from the first mutation, the secondmutation promoting a higher thermostability in the variant.
 21. Theisolated variant of claim 20, wherein the parent has a higher capabilityof using peroxide as an oxygen donor than the corresponding wild-typecytochrome P450 oxygenase heme domain.
 22. The isolated variant of claim20, having a T₅₀ higher than the wild-type cytochrome P450 domain.